CN115896048B - Recombinant human Cu, zn-SOD with high enzyme activity and good stability, and preparation method and application thereof - Google Patents

Abstract

The invention relates to recombinant human Cu, zn-SOD with high enzyme activity and good stability, a preparation method and application thereof, wherein the amino acid sequence of a coding gene is shown as SEQ ID NO. 3, the nucleotide sequence is shown as SEQ ID NO. 2, the enzyme activity of the recombinant human Cu, zn-SOD provided by the invention is about one time higher than that of natural human Cu, zn-SOD, and the enzyme activity stability is higher than that of natural human Cu, zn-SOD.

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 to act in the process of scavenging active oxygen free radicals in the body, and has good preventive and therapeutic effects on aging and other diseases caused by free radicals.

There are 4 types of superoxide dismutase currently known: 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, zn-SOD widely exist in cytoplasm, chloroplasts and catalase bodies of most eukaryotes such as mammals, fungi and the like, and are the superoxide dismutase which is most widely distributed in nature and is the most widely used at present.

At present, most superoxide dismutase (SOD) products mainly originate from animal blood, viscera and the like, and have low purity and low yield due to limited raw materials, difficult purification and other reasons; especially, with epidemic situations of malignant infectious diseases such as mad cow disease, avian influenza, foot-and-mouth disease and SARS transmitted by animals, the risk of producing animal-derived blood products is increased; in addition, the increase in product purity requirements also increases production costs. With the development of genetic engineering technology, SOD genes from different sources such as microorganisms, plants, animals and the like are cloned and expressed. However, the recombinant SOD has problems such as existence of inactive inclusion bodies, low activity, easy inactivation, and poor heat resistance.

The SOD is obtained by genetic engineering means, mainly comprising fusion expression with other proteins, point mutation of human SOD, screening of high-performance SOD from other species, and the like.

CN103789278A discloses a novel PTD4-Cu, zn-SOD fusion protein and a preparation method thereof, and the novel PTD4-Cu, zn-SOD fusion protein has very high cell membrane penetrating efficiency, so that Cu, zn-SOD which cannot pass through cell membranes originally is transduced to cell membranes to exert biological functions.

CN102526712B discloses an application of SOD-TAT fusion protein in preparing a medicament for preventing and treating radiation injury. The SOD-TAT not only can eliminate extracellular free radicals, but also can eliminate intracellular free radicals, in addition, the SOD-TAT can also cross the blood brain barrier, and the depth and the breadth of the radiation protection effect are far stronger than those of the existing clinically used radiation protection agents.

CN101603048B discloses a prokaryotic expression method of fungal cu_zn-SOD without molecular chaperones. CN104450632 discloses a group of amino acid sequences capable of improving the heat-resistant temperature and the thermal stability of SOD and application thereof. It is a group of N-terminal amino acid sequences derived from 13 specific thermophilic Fe/Mn-SOD of Geobacillus genus.

CN101525600B discloses a method for increasing the yield of recombinant human Cu, zn-SOD active protein. The invention makes the ratio of recombinant protein in the upper clear liquid of schizochytrium to the total expression of recombinant protein increased from 40% to 80% by mutating the cysteine at the 6 th and 111 th positions into serine, and the specific activity of the mutant rhCu, zn-SOD purified from the upper clear liquid is equivalent to that of the non-mutated rhCu, zn-SOD purified from the upper clear liquid. However, the patent does not address the stability of the product.

In view of this, there is still a need in the art to continue to study recombinant human Cu, 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 simultaneously provides a preparation method and application thereof.

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

the invention provides recombinant human Cu, zn-SOD, and 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 of which is shown as SEQ ID NO. 2

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

(1) Codon optimization is carried out by taking amino acid sequences of human Cu and Zn-SOD as templates to obtain a nucleotide sequence SEQ ID NO. 1, then complete gene synthesis is carried out to obtain nucleic acid fragments for encoding the human Cu and Zn-SOD, and an expression vector of genes is constructed and transformed into host cells to obtain an expression strain.

(2) Upstream primer selection: 5'-TAATACGACTCACTATAGGG-3', downstream primer 5'-GCTGAGCAATAACTAGC-3', error-prone PCR using the expression vector of the gene described in step (1) as a template; constructing an expression vector of the mutant gene, converting the expression vector into host cells, constructing a mutant library, and screening positive mutation to obtain strains;

(3) After the strain is activated, in LB culture medium inoculated according to the proportion of 10%, IPTG is used for inducing the target protein to express, and thalli are collected;

(4) And (3) crushing the bacterial 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, zn-SOD.

As a further improvement of the invention, the nucleic acid fragment encoding the human Cu, zn-SOD in the step (1) is connected to a pET30a (+) plasmid through Nde I and Kpn I restriction sites, and the pET30a (+) -SOD is transferred into BL21 (DE 3) competence to obtain an escherichia coli expression strain for expressing the human Cu, zn-SOD.

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

In a final aspect the invention provides the use of 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 stability of the enzyme activity of the recombinant human Cu, zn-SOD obtained by the invention is higher than that of natural human Cu, zn-SOD.

Drawings

In order to more clearly illustrate the embodiments of the present invention, the following brief description will be given of the drawings.

FIG. 1 is an electrophoresis chart of a whole fermentation strain and a purified sample;

m: protein Marker,1: natural Cu, zn-SOD whole bacteria, 2: natural Cu, zn-SOD bacteria breaking precipitation, 3: natural Cu, zn-SOD bacterial-destroying supernatant, 4: affinity purification of natural Cu and Zn-SOD, 5: ion exchange purification of natural Cu and Zn-SOD, 6: BSA,7: mutant Cu, zn-SOD whole bacteria, 8: mutant Cu, zn-SOD precipitation, 9: mutant Cu, zn-SOD supernatant, 10: mutant Cu, zn-SOD affinity purification, 11: mutation search 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 will be clearly and completely described below in connection with specific embodiments.

EXAMPLE 1 expression construction of native human Cu, zn-SOD E.coli

The amino acid sequences of human Cu and Zn-SOD (UniProtKB/Swiss-Prot: P00441.2) are taken as templates for codon optimization, and the following gene sequences are obtained:

(SEQ ID NO: 1), entrusting Jin Weizhi to synthesize a gene sequence, connecting the gene sequence to pET30a (+) through Nde I and Kpn I enzyme cutting sites to obtain pET30a (+) -SOD, transferring the pET30a (+) -SOD into BL21 (DE 3) competence, and obtaining the escherichia coli expression strain for expressing human Cu, zn-SOD.

EXAMPLE 2 Gene mutation

Error-prone PCR was performed using pET30a (+) -SOD as template, upstream primer: 5'-TAATACGACTCACTATAGGG-3', downstream primer 5'-GCTGAGCAATAACTAGC-3'.

The PCR system is as follows: 2X Taq PCR Master Mix. Mu.l, 1. Mu.l of plasmid template, 1. Mu.l of upstream primer, 1. Mu.l of downstream primer, 1. Mu.l of manganese chloride solution, and 50. Mu.l of ddH2O were added.

Error-prone PCR cycling procedure, namely dropping PCR, pre-denaturing for 5min at 95 ℃, and cycling for 1 time; denaturation at 95℃for 30s, denaturation at 58℃for 30s, denaturation at 72℃for 100s, a decrease in annealing temperature of 1℃per cycle, cycle 5 times; denaturation at 95℃for 30s, denaturation at 55℃for 30s, denaturation at 72℃for 100s, 25 cycles; extending at 72 ℃ for 10min, and circulating for 1 time.

EXAMPLE 3 super competent preparation and construction of mutant libraries

The obtained target gene containing a certain mutation rate is subjected to digestion and recovery by Nde I and Kpn I and then is connected with a vector pET30a (+), and a connection product is added into E.coli Bl21 competent cells which are taken out of ice in advance and melted, so that super competence with higher conversion efficiency is prepared according to the operation instruction of the super competence preparation kit. The resurrected bacterial solution is coated on LB solid medium containing kanamycin, cultured for 30min at 37 ℃, and the culture dish is inverted for overnight culture, so as to construct mutant library with higher transformant quantity.

EXAMPLE 4 induced expression and Positive mutation screening of monoclonal

Colonies on the solid medium were picked one by one into 96-well bacterial plates (each well containing 200. Mu.l of LB medium to which antibiotics had been added) and cultured at 200rpm for 12-16 hours at 37 ℃; the bacterial solution in each well was then transferred to a new 96-well plate (each well containing 500. Mu.l of LB medium to which antibiotics had been added) at an inoculum size of 2%, and cultured for 6 hours, followed by addition of IPTG for induction expression. Centrifugally collecting the induced thalli, adding 40 cell lysate into each hole, and standing at 37 ℃ for 1-2h to crack the thalli; the resulting mixture was centrifuged at 12000rpm at 4℃for 10 minutes, and the supernatant was collected, and the enzyme activity was measured as in example 5, followed by screening for positive mutations.

Mixing the rest bacterial liquid with glycerol at a ratio of 7:3, and freezing at-80deg.C to obtain seed.

Example 5 enzyme Activity assay and screening

1. Solution

And (3) solution A: 0.01mol/L Tris buffer solution (Tris-HCl) pH8.20;

and (2) liquid B: 4.5mmol/L pyrogallol hydrochloric acid solution.

2. Pyrogallol autoxidation rate determination

2.35mL of solution A, 2.00mL of distilled water and 0.15mL of solution B are sequentially added into a 10mL colorimetric tube at about 25 ℃. The solution B was added and immediately mixed and poured into a cuvette, and the absorbance at 325nm was measured at the beginning and after 1min, respectively, and the difference between the two was the pyrogallol autoxidation rate DeltaA 325 (min-1).

3. Sample enzyme activity assay

Measurement of the rate of inhibition of the self-oxidation of the pyrogallol by the SOD enzyme solution the enzyme solution is added according to the step 2 to ensure that the rate of inhibition of the self-oxidation of the pyrogallol is about 1/2 delta A325 (min-1),

TABLE 1SOD Activity assay sample addition Table

4. Result calculation

U/mL-SOD enzyme activity unit;

delta A325-rate of pyrogallol autoxidation;

delta A' 325-sample solution or SOD enzyme solution inhibits the rate of pyrogallol autoxidation;

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

d, dilution factor of enzyme solution or sample solution;

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

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

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 the primary screening of the directed evolution of the target genes. In order to further determine the expression and activity of the mutant, the mutant obtained by primary screening is subjected to shake flask expansion culture, thalli are collected, and the thalli are crushed by ultrasound to obtain the target protein; the enzyme activity was measured as in example 5, and protein expression was detected by SDS-PAGE. Mutants with high enzyme activity were further identified. Finally, the mutants 2-A6, 8-F11, 14-D3, 14-E2 and 20-B3 were determined to have the highest enzyme activities. After that, the stability of SOD obtained by each mutant was studied, and only 8-F11 mutants were good in stability, so that the following examples were only 8-F11.

EXAMPLE 7 high expression mutant plasmid extraction and sequencing

The high-enzyme activity mutant 8-F11 obtained by re-screening is coated on LB solid medium containing kanamycin, and after 30min of culture at 37 ℃, the culture dish is inverted for overnight culture. The monoclonal was picked up and inoculated into LB-containing shake flasks, cultured overnight at 37℃and 180 rpm. Plasmids were extracted using the Soxhaust plasmid extraction kit.

1. Taking 5mL of bacterial liquid, centrifuging at 12000rpm for 1min, and sucking the supernatant as much as possible

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

3. 250 mu L of solution II is added into the centrifuge tube, and the tube is gently turned up and down for 6-8 times to fully lyse the thalli.

4. 350. Mu.L of solution III was added to the centrifuge tube, immediately and gently turned upside down 6-8 times, and thoroughly mixed, at which time white flocculent precipitate appeared. Centrifuge at 12000rpm for 10min, carefully transfer the supernatant to another clean centrifuge tube with a pipette, and minimize aspiration of pellet.

5. Adding the supernatant obtained in the previous step into an adsorption column (the adsorption column is added into a collecting pipe), standing at room temperature for 2min, centrifuging at 12000rpm for 1min, pouring out the waste liquid in the collecting pipe, and putting the adsorption column back into the collecting pipe.

6. 600. Mu.L of rinse liquid I (absolute ethanol was previously added) was added to the column, centrifuged at 12000rpm for 1min, the waste liquid was discarded, and the column was placed in a collection tube.

7. 700. Mu.L of rinse II (absolute ethanol was previously added) was added to the column, centrifuged at 12000rpm for 1min, the waste liquid was discarded, and the column was placed in a collection tube.

8. 500. Mu.L of rinsing liquid II was added to the column, centrifuged at 12000rpm for 1min, the waste liquid was discarded, and the column was placed in a collection tube.

Centrifuging at 9.12000rpm for 2min, placing the adsorption column in room temperature or 50deg.C incubator for several minutes to remove residual rinse solution

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

The extracted plasmid was subjected to Jin Weizhi sequencing to obtain the mutated gene sequence as follows (SEQ ID NO: 2):

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

by comparison with the amino acid sequences of native human Cu, zn-SOD, it was found that valine at position 15 was mutated to glycine and phenylalanine at position 51 was mutated to valine as determined by sequencing.

EXAMPLE 8 shake flask amplification culture of high expression mutant

After activating the 8-F11 mutant strain with high enzyme activity, inoculating the strain into 30ml of LB culture medium at a ratio of 1 per mill, and culturing at 37 ℃ and 180rpm for overnight. Then, the cells were inoculated into 100ml of LB medium at a ratio of 10% and cultured for 3 hours, followed by induction with 0.5mM IPTG for 6 hours, and then 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 1:20, and then sonicated. The supernatant was collected by centrifugation at 12000g for 10min and filtered through a 0.45 μm filter.

2. Affinity chromatography

And (3) solution A: 20mM Tris,500mM NaCl,20mM imidazole, pH8.0

And (2) liquid B: 20mM Tris,500mM NaCl,500mM imidazole, pH8.0

The supernatant in the step 1 is loaded at a flow rate of 1ml/min by using a GE Ni Sepharose 6FF 5mL pre-loaded column and balancing with A solution; then washing impurities with the solution A, wherein the volume is 25ml; eluting with 50% B solution, and collecting eluting peak to obtain the final product.

3. Ion exchange

And (3) solution A: 20mM Tris, pH8.0

And (2) liquid B: 20mM Tris,1M NaCl,pH8.0

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

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

Test example 1

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

The samples from each of the above steps were subjected to SDS-PAGE, and the results are shown in FIG. 1. The graph shows that (1) the expression quantity of mutant SOD and natural SOD is not different, and can reach more than 60% of the total mycoprotein; (2) SOD is mainly expressed in a soluble form, and the target protein in the sediment has a small proportion; (2) The purity of SOD is very high, no obvious impurity protein band exists, and the purity is more than 98%.

Determination of enzyme Activity

The enzyme activities of the native 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 the specific activity was increased by nearly one time after mutation.

Test example 2 stability of purified SOD

The SOD pure product obtained in test example 1 was sterilized by filtration, and then packaged in 1.5ml sterile EP tubes, 100. Mu.l/tube, and stored at room temperature, 4 ℃, -20℃and-80℃respectively, and the stability of the sample was examined. The data are given in the following Table (U/mg):

from the data, the enzyme activity of the natural SOD at room temperature and 4 ℃ is gradually reduced along with the extension of the storage time; the enzyme activity of the mutant SOD has no obvious change with time. Both the enzyme activities are stable when stored at-20 ℃ and-80 ℃.

Finally, it should be noted that: the above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the 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 scheme described in the foregoing embodiments can be modified or some of the technical features thereof can be replaced by equivalents; such modifications and substitutions do not depart from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims (3)

1. The 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 according to claim 1, wherein the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 2.

3. Use of the recombinant human Cu, zn-SOD of claim 1 in the preparation of a medical or cosmetic product.