Engineering strain for soluble expression of human Cu / Zn superoxide dismutase as well as construction method and application of engineering strain

Abstract

The invention discloses an engineering strain for soluble expression of human Cu / Zn superoxide dismutase as well as a construction method and application of the engineering strain, and relates to the technical field of biology. The construction method of the engineering strain comprises the following steps: cloning a coding gene of the human Cu / Zn superoxide dismutase into an expression vector to construct a recombinant plasmid; transforming the recombinant plasmid into escherichia coli competent cells to construct the engineering strain; the nucleotide sequence of the coding gene is as shown in SEQ ID NO. 2. The invention provides a new engineering strain for improving the expression abundance of the human Cu / Zn superoxide dismutase in escherichia coli and the proportion of a soluble expression part, and has important industrial application value.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly to an engineered strain for soluble expression of human Cu / Zn superoxide dismutase, a construction method thereof, and an application thereof. Background Art

[0002] Superoxide Dismutase (SOD) is an important class of antioxidant enzymes that can catalyze the dismutation of superoxide anion radicals (O 2- ) into hydrogen peroxide (H 2 O 2 ) and oxygen (O 2 ), thereby protecting cells from damage by Reactive Oxygen Species (ROS). SOD is widely distributed in organisms and is divided into Cu / Zn superoxide dismutase, manganese superoxide dismutase, and iron superoxide dismutase according to different metal cofactors. Among them, human Cu / Zn superoxide dismutase has broad application prospects in the fields of medicine, cosmetics, etc. due to its high efficiency, stability, and good compatibility with the human body.

[0003] Currently, the expression level of human Cu / Zn superoxide dismutase expressed in Escherichia coli is generally low, and most of it is inclusion bodies, and active protein can only be obtained after denaturation and renaturation, which affects the industrialization of human Cu / Zn superoxide dismutase. Therefore, it is necessary to develop new engineered strains to increase the abundance of human Cu / Zn superoxide dismutase expressed in Escherichia coli and the proportion of the soluble expression part. Summary of the Invention

[0004] The object of the present invention is to provide an engineered strain for soluble expression of human Cu / Zn superoxide dismutase, a construction method thereof, and an application thereof, so as to solve the problems existing in the above-mentioned prior art. The present invention provides a new engineered strain to increase the abundance of human Cu / Zn superoxide dismutase expressed in Escherichia coli and the proportion of the soluble expression part, and has important industrial application value.

[0005] To achieve the above object, the present invention provides the following solution:

[0006] The present invention provides a construction method of an engineered strain for soluble expression of human Cu / Zn superoxide dismutase, comprising the following steps:

[0007] Clone the coding gene of human Cu / Zn superoxide dismutase into an expression vector to construct a recombinant plasmid;

[0008] Transform the recombinant plasmid into Escherichia coli competent cells to construct the engineered strain;

[0009] The nucleotide sequence of the coding gene is as shown in SEQ ID NO.2.

[0010] Further, the expression vector is pET28a vector.

[0011] Further, the coding gene is cloned between the NcoI and XhoI restriction enzyme cleavage sites of the expression vector.

[0012] Further, the coding gene is cloned between the NdeI and XhoI restriction enzyme cleavage sites of the expression vector.

[0013] Further, the coding gene is cloned between the EcoRI and XhoI restriction enzyme cleavage sites of the expression vector.

[0014] Further, the recombinant plasmid is transformed into the Escherichia coli competent cells by heat shock method.

[0015] The present invention also provides an engineered strain for soluble expression of human Cu / Zn superoxide dismutase constructed according to the above construction method.

[0016] The present invention also provides the application of the above engineered strain in the fermentative production of human Cu / Zn superoxide dismutase.

[0017] The present invention also provides a method for fermentative production of human Cu / Zn superoxide dismutase, which includes the steps of obtaining bacterial cells after fermentatively culturing the above engineered bacteria, disrupting the bacterial cells, and extracting the human Cu / Zn superoxide dismutase.

[0018] Further, during the fermentative culture process, isopropyl-β-D-thiogalactoside is used for protein induction expression.

[0019] The present invention discloses the following technical effects:

[0020] After codon optimization of the coding gene of human Cu / Zn superoxide dismutase, the present invention obtains a gene sequence with improved translation initiation speed and capable of promoting correct folding of the tertiary structure. The optimized gene sequence is cloned between the NcoI / XhoI restriction enzyme cleavage sites, NdeI / XhoI restriction enzyme cleavage sites and EcoRI / XhoI restriction enzyme cleavage sites of the pET28a vector respectively, and transformed into Escherichia coli, thereby constructing three engineered strains capable of realizing soluble expression of human Cu / Zn superoxide dismutase. The present invention provides new engineered strains for improving the expression abundance and the proportion of soluble expression part of human Cu / Zn superoxide dismutase in Escherichia coli, and has important industrial application value. Description of the Drawings

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained based on these drawings.

[0022] Figure 1 It is a comparison diagram of the original sequence and the optimized sequence;

[0023] Figure 2 It is a schematic diagram of the local structure of mRNA before optimization;

[0024] Figure 3 It is a schematic diagram of the local structure of mRNA after optimization;

[0025] Figure 4 It is a protein electrophoresis result diagram of the pET28a-SEQ 03 strain;

[0026] Figure 5 It is a protein electrophoresis result diagram of the pET28a-SEQ 04 strain;

[0027] Figure 6 It is a protein electrophoresis result diagram of the pET28a-SEQ 05 strain. Detailed Embodiments

[0028] Now, various exemplary embodiments of the present invention will be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0029] It should be understood that the terms described in the present invention are only for describing specific embodiments and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Any intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded from the range.

[0030] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein can also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0031] Without departing from the scope or spirit of the present invention, various improvements and variations can be made to the specific embodiments of the specification of the present invention, which are obvious to those skilled in the art. Other embodiments obtained from the specification of the present invention are obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0032] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0033] The pET28a vector used in the following examples was purchased from Boshang Biotechnology (Shanghai) Co., Ltd., and the competent cells of Escherichia coli BL21(DE3) were purchased from Beyotime Biotechnology Co., Ltd.

[0034] Example 1

[0035] 1. Artificial optimization of nucleic acid sequence

[0036] To improve the abundance of human Cu / Zn superoxide dismutase expressed in Escherichia coli and the proportion of the soluble expression part, the original cDNA (SEQ ID NO.1) was rationally optimized:

[0037] First, by replacing degenerate codons, the hairpin structure at the translation start point was removed to increase the translation initiation rate; second, by replacing degenerate codons at appropriate positions, the minimum free energy of the mRNA structure was increased, the translation rate after translation initiation was reduced, and the correct folding of the tertiary structure was promoted. The optimized cDNA sequence is shown in SEQ ID NO.2.

[0038] The comparison results between the original sequence and the optimized sequence are as Figure 1 shown; the local structure of the mRNA before optimization is as Figure 2 shown, the minimum free energy of the structure is -143.89 KJ / mol, and there is an obvious hairpin structure; the local structure of the mRNA after optimization is as Figure 3 shown, the minimum free energy of the structure is -146.10 KJ / mol, and there is no hairpin structure.

[0039] The original cDNA sequence (SEQ ID NO.1):

[0040] ATGGCGACGAAGGCCGTGTGCGTGCTGAAGGGCGACGGCCCAGTGCAGGGCATCATCAATTTCGAGCAGAAGGAAAGTAATGGACCAGTGAAGGTGTGGGGAAGCATTAAAGGACTGACTGAAGGCCTGCATGGATTCCATGTTCATGAGTTTGGAGATAATACAGCAGGCTGTACCAGTGCAGGTCCTCACTTTAATCCTCTATCCAGAAAACACGGTGGGCCAAAGGATGAAGAGAGGCATGTTGGAGACTTGGGCAATGTGACTGCTGACAAAGATGGTGTGGCCGATGTGTCTATTGAAGATTCTGTGATCTCACTCTCAGGAGACCATTGCATCATTGGCCGCACACTGGTGGTCCATGAAAAAGCAGATGACTTGGGCAAAGGTGGAAATGAAGAAAGTACAAAGACAGGAAACGCTGGAAGTCGTTTGGCTTGTGGTGTAATTGGGATCGCCCAATAA

[0041] Optimized cDNA sequence (SEQ ID NO.2):

[0042] ATGGCTACAAAGGCTGTATGTGTACTGAAGGGTGACGGACCAGTACAGGGCATCATCAATTTCGAGCAGAAGGAAAGTAATGGACCAGTGAAGGTGTGGGGAAGCATTAAAGGACTCACTGAAGGTCTGCATGGATTCCATGTCCATGAGTTCGGAGATAATACAGCAGGCTGTACCAGTGCTGGCCCTCACTTTAATCCTCTTTCCCGTAAACACGGTGGGCCAAAGGATGAGGAACGTCATGTAGGAGACTTGGGCAATGTGACTGCTGACAAAGATGGTGTGGCCGATGTGTCTATTGAAGATTCTGTGATCTCACTCTCAGGGGACCATTGCATCATTGGCCGGACACTGGTGGTCCATGAAAAAGCAGATGACTTGGGCAAAGGTGGAAATGAAGAAAGTACAAAGACAGGAAACGCTGGAAGTCGTTTGGCTTGTGGTGTAATTGGGATCGCCCAATAA

[0043] 2. Construction of expression vector

[0044] The optimized cDNA sequence (SEQ ID NO.2) was submitted to Boshang Biotechnology (Shanghai) Co., Ltd. for gene synthesis and cloned into the NcoI / XhoI restriction sites, NdeI / XhoI restriction sites and EcoRI / XhoI restriction sites of the pET28a vector respectively, and the recombinant plasmids pET28a-SEQ 03, pET28a-SEQ 04 and pET28a-SEQ 05 were obtained accordingly.

[0045] 3. Construction of expression strains

[0046] The synthesized recombinant plasmids pET28a-SEQ 03, pET28a-SEQ 04 and pET28a-SEQ 05 were transformed into Escherichia coli BL21(DE3) competent cells by heat shock method respectively, spread on LB plates containing kanamycin, and cultured at 37 °C for overnight (about 16 hours) statically. The single colonies picked were the expression strains.

[0047] 4. Expression verification

[0048] From the plates transformed with the three plasmids, pick one single colony each and inoculate them into test tubes containing 3 mL of LB liquid medium with kanamycin. Incubate them on a shaker at 220 rpm and 37 °C for 4.5 hours. Then transfer 1 mL to 50 mL of LB medium and incubate for about 4 hours until the OD 600 reaches around 1.5. Add isopropyl-β-D-thiogalactoside (IPTG) at a final concentration of 0.2 mM and adjust the temperature to 28 °C for overnight induction of expression.

[0049] 5. SDS-PAGE Analysis of Expression Results

[0050] After overnight expression, centrifuge the cells at 4500 rpm for 5 min to collect the cell pellet. Add purified water to the cell pellet until the OD 600 ≈20, and then disrupt the cells by sonication. The sonicated cell lysate is divided into whole cell, supernatant, and pellet fractions to prepare SDS-PAGE samples respectively. The SDS-PAGE results are as Figures 4 - 6 shown.

[0051] The SDS-PAGE results show that the protein expression abundances of the three strains are all good, and a relatively high proportion of them are soluble expressions. Among them, the pET28a-SEQ 03 strain is basically all soluble expression, the pET28a-SEQ 04 strain has more than about 3 / 4 soluble expression, and the pET28a-SEQ 05 strain has more than about 1 / 2 soluble expression.

[0052] The amino acid sequences of the human Cu / Zn superoxide dismutase expressed by the pET28a-SEQ 03 strain, pET28a-SEQ 04 strain, and pET28a-SEQ 05 strain are shown as SEQ ID NO.3 - 5 respectively.

[0053] SEQ ID NO.3:

[0054] MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ LEHHHHHH *.

[0055] SEQ ID NO.4:

[0056] MGSSHHHHHHSSGLVPRGSHMATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ*。

[0057] SEQ ID NO.5:

[0058] MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSEF MATKAVCVLKGDGPVQGIINFEQKESNGPVKVWGSIKGLTEGLHGFHVHEFGDNTAGCTSAGPHFNPLSRKHGGPKDEERHVGDLGNVTADKDGVADVSIEDSVISLSGDHCIIGRTLVVHEKADDLGKGGNEESTKTGNAGSRLACGVIGIAQ*。

[0059] 6. Enzyme activity assay

[0060] CuSO with a final concentration of 4 mM was added to the broken bacterial solution respectively 4 for activation. The activation temperature was room temperature (20 - 30 °C) and the activation time was 15 hours. After activation, the enzyme solution was centrifuged at 12,000 rpm for 5 min, and the supernatant was taken for activity assay. The activity assay was carried out according to the method of national standard GBT5009.171 - 2003.

[0061] The results showed that the specific activity of wet cells measured for the pET28a - SEQ 03 strain was: 485,000 U / g wet cells, for the pET28a - SEQ04 strain was 53,900 U / g wet cells, and for the pET28a - SEQ 05 strain was 43,300 U / g wet cells.

[0062] The above - described embodiments are only descriptions of the preferred embodiments of the present invention, and do not limit the scope of the present invention. Without departing from the design spirit of the present invention, various deformations and improvements made by those of ordinary skill in the art to the technical solutions of the present invention shall fall within the protection scope determined by the claims of the present invention.

Claims

1. A method for constructing an engineered strain that soluble expresses human Cu / Zn superoxide dismutase, characterized in that: The following steps are involved: The coding gene of human Cu / Zn superoxide dismutase was cloned into an expression vector to construct a recombinant plasmid; Transforming the recombinant plasmid into competent Escherichia coli cells to construct the engineered strain; The nucleotide sequence of the coding gene is shown in SEQ ID NO.

2.

2. The construction method according to claim 1, characterized in that: The expression vector is pET28a vector.

3. The construction method according to claim 2, characterized in that: The coding gene is cloned between the NcoI and XhoI restriction sites of the expression vector.

4. The construction method according to claim 2, characterized in that: The coding gene is cloned between the NdeI and XhoI restriction sites of the expression vector.

5. The construction method according to claim 2, characterized in that: The coding gene is cloned between the EcoRI and XhoI restriction sites of the expression vector.

6. The construction method according to claim 1, characterized in that: The recombinant plasmid is transformed into the competent E. coli cells by heat shock method.

7. An engineered strain expressing soluble human Cu / Zn superoxide dismutase constructed according to the construction method according to any one of claims 1 to 6.

8. Use of the engineered strain as claimed in claim 7 in the fermentation production of human Cu / Zn superoxide dismutase.

9. A method for producing human Cu / Zn superoxide dismutase by fermentation, characterized in that: The method comprises the steps of fermenting and culturing the engineered bacteria as claimed in claim 7 to obtain bacterial bodies, crushing the bacterial bodies, and then extracting the human Cu / Zn superoxide dismutase.

10. The method according to claim 9, characterized in that During the fermentation and culturing process, isopropyl-β-D-thiogalactoside is used to induce protein expression.

Images