CN110776572A - Fusion protein of 7 β -HSDH enzyme and DPS - Google Patents
Fusion protein of 7 β -HSDH enzyme and DPS Download PDFInfo
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- CN110776572A CN110776572A CN201911114498.3A CN201911114498A CN110776572A CN 110776572 A CN110776572 A CN 110776572A CN 201911114498 A CN201911114498 A CN 201911114498A CN 110776572 A CN110776572 A CN 110776572A
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- C12Y101/01—Oxidoreductases acting on the CH-OH group of donors (1.1) with NAD+ or NADP+ as acceptor (1.1.1)
- C12Y101/01201—7-Beta-hydroxysteroid dehydrogenase (NADP+) (1.1.1.201)
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Abstract
The invention relates to a fusion protein of 7 β -HSDH enzyme and DPS, which mutates glycine at 159 th site of 7 β -HSDH enzyme into histidine and lysine at 165 th site into histidine, wherein the amino acid SEQUENCE of the glycine is shown as SEQUENCE NO.1, so as to obtain a 7 β -HSDH enzyme mutant, the amino acid SEQUENCE of which is shown as SEQUENCE NO.2, and the 7 β -HSDH enzyme mutant is fused and expressed with DPS protein from Thermoanaerobacter pseudethanolicus to obtain the fusion protein of the 7 β -HSDH enzyme and the DPS, compared with wild 7 β -HSDH enzyme, the thermal stability of the fusion protein of the 7 β -HSDH enzyme and the DPS is obviously improved, and the enzyme activity is improved by 70%.
Description
Technical Field
The invention relates to the technical field of fusion proteins, in particular to a fusion protein of 7 β -HSDH enzyme and DPS.
Background
Tauroursodeoxycholic acid (TUDCTUA) is a main pharmacological active ingredient in bear gall, and is mainly obtained by manually extracting bear gall at present, wherein the Tauroursodeoxycholic acid is mainly used for clinically treating diseases related to abnormal bile acid metabolism, but due to scarcity of bear gall resources and particularity of sources, the yield of the Tauroursodeoxycholic acid (TUDCA) can not meet the requirements of clinical medication.
Disclosure of Invention
The invention analyzes and compares the thermal stability and the enzymatic activity of 4 kinds of 7 β -HSDH enzymes from Ruminococcus torques, Ruminococcus gnavus, Collinlla aerofaciens and Clostridium sardiniense, carries out point mutation design on the 7 β -HSDH enzyme from the Ruminococcus gnavus, and carries out fusion expression on the 7 β -HSDH enzyme mutant and DPS protein so as to improve the activity and the thermal stability of the 7 β -HSDH enzyme and facilitate the subsequent industrial catalytic application.
A fusion protein of 7 β -HSDH enzyme and DPS is prepared through mutating the 159 th glycine and 165 th lysine of 7 β -HSDH enzyme to histidine and obtaining the 7 β -HSDH enzyme mutant with amino acid SEQUENCE shown in SEQUENCE NO.2, fusing the 7 β -HSDH enzyme mutant with DPS protein from Thermoanaerobacter pseudethanolicus to obtain the fusion protein of 7 β -HSDH enzyme and DPS.
The 7 β -HSDH enzyme is derived from the genus Ruminococcus gnavus.
The preparation method of the fusion protein of the 7 β -HSDH enzyme and the DPS comprises the steps of constructing a 7 β -HSDH enzyme mutant and the DPS protein on an escherichia coli pGEX6P-1 vector by a PCR or gene synthesis method, transforming an escherichia coli BL21(DE3) competent cell by the obtained recombinant vector, and carrying out induced expression on the recombinant protein to obtain the fusion protein of the 7 β -HSDH enzyme and the DPS.
Compared with wild 7 β -HSDH enzyme, the thermal stability and the enzyme activity of the 7 β -HSDH and DPS fusion protein obtained by the invention are obviously higher than those of the fusion protein, and the enzyme activity is improved by 70%.
Description of the drawings:
FIG. 1 is a plasmid map of pGEX6P-1-N-GST-3C-7 β -HSDH mutant.
FIG. 2 is a plasmid map of pGEX6P-1-N-GST-3C-DPS-linker-7 β -HSDH.
FIG. 3 shows the result of SDS-PAGE analysis of 7 β -HSDH enzyme (Ruminococcus torque), lane 1: 2ug of target protein and lane 2: 10ug of target protein.
FIG. 4 shows the result of SDS-PAGE analysis of 7 β -HSDH (Ruminococcus gnavus), lane 1: 2ug of the target protein, and lane 2: 10ug of the target protein.
FIG. 5 shows the results of SDS-PAGE analysis of 7 β -HSD enzyme (Collinsella aerofaciens), lane 1: 2ug of target protein and lane 2: 10ug of target protein.
FIG. 6 shows the results of SDS-PAGE analysis of 7 β -HSDH enzyme (Clostridium sardiniense), lane 1: 2ug of target protein and lane 2: 10ug of target protein.
FIG. 7 shows the SDS-PAGE analysis of the fusion protein of 7 β -HSDH enzyme and DPS, lane 1: 2ug of the target protein, and lane 2: 10ug of the target protein.
FIG. 8 shows the results of thermal stability analysis of 7 β -HSDH enzyme and 7 β -HSDH enzyme fused with DPS from 4 species of species, wherein the 7 # derived from Ruminococcus torques 7 β -HSDH enzyme is the graph shown in the figure, the 2# derived from Ruminococcus gnavus7 β -HSDH enzyme is the graph shown in the figure, the 3# derived from Collinsella aerofaciens 7 β -HSDH enzyme is the graph shown in the figure, the 4# derived from Clostridium sardiniense 7 β -HSDH enzyme is the graph shown in the figure, and the 5# derived from 7 β -HSDH enzyme fused with DPS protein is the graph shown in the figure.
FIG. 9 shows the results of the assays of the activities of the 4 species of 7 β -HSDH enzyme and 7 β -HSDH enzyme fused with DPS.
Detailed Description
The invention is described below by means of specific embodiments. Unless otherwise specified, all technical means described herein are known to those skilled in the art. Furthermore, the embodiments are to be regarded as illustrative rather than limiting the scope of the invention. The spirit and scope of the present invention are limited only by the appended claims.
Materials (I) and (II)
1.1 expression vector (1) pGEX6P-1-N-GST-3C-7 β -HSDH, the plasmid map of which is shown as 1;
(2) pGEX6P-1-N-GST-3C-DPS-linker-7 β -HSDH was constructed by the company itself, and its plasmid map is shown in FIG. 2.
1.2 competent cells: a TransT1 competent cell, BL21 competent cell.
1.3 culture medium and reagents: LB medium, IPTG inducer, L-glutamhiene (reduced), SYPROOrange dye, NADP, TUDCA, plasmid miniprep kit (TIANGEN), general agarose gel recovery kit (TIANGEN)
1.4 Experimental facility SPH-211C temperature control shaking table, AKTA-FPLC rapid protein purification liquid phase, Agilent LC-MS mass spectrum analyzer, Biometra TProfessional PCR instrument, LRH-70F biochemical incubator, MD SpectraMax Plus384 continuous wavelength microplate reader, ABI7500.
Second, method
2.17 β -HSDH enzyme point mutation design
The whole gene sequence DNA of 4 wild type 7 β -hydroxysteroid dehydrogenases (7 β -HSDH) from the species Ruminococcus torques, Ruminococcus gnavus, Collinsella aerofaciens and Clostridium sardiniense were obtained by PCR or gene synthesis, respectively.
The 7 β -HSDH amino acid SEQUENCE derived from the genus Ruminococcus gnavus is shown as SEQUENCE NO. 1. the 7 β -HSDH whole gene SEQUENCE derived from the genus Ruminococcus gnavus is subjected to point mutation design, the 159 th glycine is mutated into histidine, and the 165 th lysine is mutated into histidine, and the 7 β -HSDH enzyme (G159H, K165H) mutant SEQUENCE is obtained by a site-directed mutation method and is shown as SEQUENCE NO. 2.
2.27 β -HSDH expression vector construction
With GST as an affinity tag, a 7 β -HSDH enzyme (G159H, K165H) mutation sequence derived from the genus of Ruminococcus gnavus and a DPS protein gene derived from the genus of Thermoanaerobacter pseudolitelinus are introduced into an Escherichia coli pGEX6P-1 vector by a PCR seamless cloning method to construct a recombinant plasmid:
pGEX6P-1-N-GST-3C-7 β -HSDH and pGEX6P-1-N-GST-3C-DPS-linker-7 β -HSDH.
Meanwhile, the DNA of the whole gene sequence of 4 wild type 7 β -hydroxysteroid dehydrogenases (7 β -HSDH) from the species of Ruminococcus torques, Ruminococcus gnavus, Collinsellaerofaciens and Clostridium sardiniense is introduced into an Escherichia coli pGEX6P-1 vector to construct a recombinant plasmid pGEX6P-1-N-GST-3C-7 β -HSDH
2.37 β -HSDH protein expression
Escherichia coli BL21(DE3) is prepared, recombinant plasmids pGEX6P-1-N-GST-3C-7 β -HSDH and pGEX6P-1-N-GST-3C-DPS-linker-7 β -HSDH are transformed into Escherichia coli BL21(DE3) competence, and the target protein is obtained from 2L liquid culture medium according to the culture temperature of 15 ℃, the IPTG final concentration of 0.5mM and the induction time of 16 h.
2.47 β -HSDH enzyme and DPS fusion protein purification and SDS-PAGE electrophoretic analysis
The 4 kinds of 7 β -HSDH enzyme, 7 β -HSDH enzyme mutant and DPS fusion protein were purified by affinity chromatography, and the expression and purification effects of the 4 kinds of 7 β -HSDH enzyme, 7 β -HSDH enzyme mutant and DPS fusion protein were analyzed by SDS-PAGE electrophoresis, as shown in FIG. 3, FIG. 4, FIG. 5, FIG. 6 and FIG. 7.
2.57 β -HSDH enzyme and DPS fusion protein thermostability analysis
The thermal stability analysis of 7 β -HSDH enzyme, 7 β -HSDH enzyme and DPS fusion protein was performed using SYPRO Orange dye, protein purification buffer, target protein and SYPRO Orange dye were added to a 96-well microplate, and the program was set at 95 ℃ for 2min, 25 ℃ for 2min and the temperature ramp rate was set at 1 ℃ per minute, as shown in FIG. 8.
2.67 β -HSDH enzyme and DPS fusion protease Activity assay
The enzyme activity of the obtained 7 β -HSDH enzyme, 7 β -HSDH enzyme and DPS fusion protein was analyzed, and a buffer, a target protein, NADP and TUDCA were added to a 96-well reaction plate to measure the change in absorbance at 340 nM.
Third, result analysis
3.1 analysis of protein expression level
Through experimental determination, under the same expression conditions of the four 7 β -HSDH enzymes and the 7 β -HSDH enzyme mutant and the DPS fusion protein, the expression amounts of the proteins are different, the expression amount of the 7 β -HSDH enzyme and the DPS fusion protein is the highest, and the expression amount of the 7 β -HSDH enzyme from the genus of Ruminococcus gnavus is the second expression amount, which is shown in Table 1.
3.2 analysis of thermal stability results
According to experimental determination, the Tm average value of the fusion protein of the 7 β -HSDH enzyme and the DPS is 64.43 ℃, the Tm average value of the 7 β -HSDH enzyme derived from the genus Ruminococcus torques is 55.01, the Tm average value of the 7 β -HSDH enzyme derived from the genus Ruminococcus gnavus is 55.85, the Tm average value of the 7 β -HSDH enzyme derived from the genus Collinella aerofacies is 54.58, the Tm average value of the 7 β -HSDH enzyme derived from the genus Clostridia sardiniense is 54.07, and the thermal stability of the 7 β -HSDH enzyme and the DPS fusion protein is improved by 20 percent compared with that of the 7 β -HSDH enzyme of the 4 species, as shown in Table 1.
3.3 analysis of enzyme Activity results
According to experimental determination, the activity of 7 β -HSDH enzyme derived from the genus Ruminococcus torques is 41.1U/mg, the activity of 7 β -HSDH enzyme derived from the genus Ruminococcus gnavus is 68.8U/mg, the activity of 7 β -HSDH enzyme derived from the genus Collinsellaerofacies is 30.5U/mg, the activity of 7 β -HSDH enzyme derived from the genus Clostridia sardiniense is 34.1U/mg, the activity of fusion protease of 7 β -HSDH enzyme and DPS is 120.8U/mg, the activity of fusion protease of 7 β -HSDH enzyme and DPS is obviously higher than that of 7 β -HSDH enzyme derived from the 4 species, and the enzyme activity is improved by 70%, see Table 1 and figure 9.
TABLE 1 comparison of expression level, thermostability, and enzyme Activity of each protein
The foregoing is a more detailed description of the invention in connection with specific preferred embodiments and it is not intended that the invention be limited to these specific details. For those skilled in the art to which the present invention pertains, the architecture form can be flexible and varied without departing from the concept of the present invention, and a series of products can be derived. But rather a number of simple derivations or substitutions are made which are to be considered as falling within the scope of the invention as defined by the appended claims.
Sequence listing
<120> fusion protein of 7 β -HSDH enzyme and DPS
<160>2
<170>SIPOSequenceListing 1.0
<210>1
<211>263
<212>PRT
<213>Ruminococcus gnavus
<400>1
Met Thr Leu Arg Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Arg Leu Ala Lys Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Lys Glu Leu
35 40 45
Gly Glu Glu Leu Lys Asn Thr Tyr Glu Ile Asp Tyr Lys Val Val Lys
50 55 60
Ala Asp Phe Ser Leu Pro Asp Ala Thr Asp Lys Ile Phe Ala Ala Thr
65 70 75 80
Glu Asn Leu Asp Met Gly Phe Met Ala Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Met Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala Gly Lys
145 150 155 160
Ala Phe Ile Leu Lys Met Thr Glu Ala Val Ala Cys Glu Thr Glu Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Thr Ala Gln Thr Pro Glu Glu Val Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ser Gly Glu Arg Asn Lys Ala Ser
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Asp Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Gln Glu
260
<210>2
<211>263
<212>PRT
<213>Ruminococcus gnavus
<400>2
Met Thr Leu Arg Glu Lys Tyr Gly Glu Trp Gly Ile Ile Leu Gly Ala
1 5 10 15
Thr Glu Gly Val Gly Lys Ala Phe Cys Glu Arg Leu Ala Lys Glu Gly
20 25 30
Met Asn Val Val Met Val Gly Arg Arg Glu Glu Lys Leu Lys Glu Leu
35 40 45
Gly Glu Glu Leu Lys Asn Thr Tyr Glu Ile Asp Tyr Lys Val Val Lys
50 55 60
Ala Asp PheSer Leu Pro Asp Ala Thr Asp Lys Ile Phe Ala Ala Thr
65 70 75 80
Glu Asn Leu Asp Met Gly Phe Met Ala Tyr Val Ala Cys Leu His Ser
85 90 95
Phe Gly Lys Ile Gln Asp Thr Pro Trp Glu Lys His Glu Ala Met Ile
100 105 110
Asn Val Asn Val Val Thr Phe Met Lys Cys Phe Tyr His Tyr Met Lys
115 120 125
Ile Phe Ala Ala Gln Asp Arg Gly Ala Val Ile Asn Val Ser Ser Met
130 135 140
Thr Gly Ile Ser Ser Ser Pro Trp Asn Gly Gln Tyr Gly Ala His Lys
145 150 155 160
Ala Phe Ile Leu His Met Thr Glu Ala Val Ala Cys Glu Thr Glu Lys
165 170 175
Thr Asn Val Asp Val Glu Val Ile Thr Leu Gly Thr Thr Leu Thr Pro
180 185 190
Ser Leu Leu Ser Asn Leu Pro Gly Gly Pro Gln Gly Glu Ala Val Met
195 200 205
Lys Thr Ala Gln Thr Pro Glu Glu Val Val Asp Glu Ala Phe Glu Lys
210 215 220
Leu Gly Lys Glu Leu Ser Val Ile Ser Gly Glu Arg Asn Lys Ala Ser
225 230 235 240
Val His Asp Trp Lys Ala Asn His Thr Glu Asp Asp Tyr Ile Arg Tyr
245 250 255
Met Gly Ser Phe Tyr Gln Glu
260
Claims (3)
1. A fusion protein of 7 β -HSDH enzyme and DPS is characterized in that glycine at 159 th site of 7 β -HSDH enzyme with an amino acid SEQUENCE shown as SEQUENCE NO.1 is mutated into histidine, lysine at 165 th site is mutated into histidine to obtain a 7 β -HSDH enzyme mutant with an amino acid SEQUENCE shown as SEQUENCE ID NO.2, and the 7 β -HSDH enzyme mutant and DPS protein from Thermoanaerobacter pseudethanolicus are subjected to fusion expression to obtain the fusion protein of the 7 β -HSDH enzyme and the DPS.
2. The fusion protein of 7 β -HSDH enzyme and DPS according to claim 1, wherein said 7 β -HSDH enzyme is derived from the genus Ruminococcus gnavus.
3. The method for preparing the fusion protein of 7 β -HSDH enzyme and DPS as claimed in claim 1 or 2, wherein the 7 β -HSDH enzyme mutant and DPS protein are constructed on the vector of Escherichia coli pGEX6P-1 by PCR or gene synthesis, the obtained recombinant vector is transformed into competent cells of Escherichia coli BL21(DE3), and the fusion protein of 7 β -HSDH enzyme and DPS is obtained by induced expression of the recombinant protein.
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| CN111944775A (en) * | 2020-08-21 | 2020-11-17 | 苏州引航生物科技有限公司 | Modified 7 beta-hydroxysteroid dehydrogenase and application thereof |
| CN112029740A (en) * | 2020-09-15 | 2020-12-04 | 江西邦泰绿色生物合成生态产业园发展有限公司 | 7 beta hydroxysteroid dehydrogenase mutant and application thereof |
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| CN111944775B (en) * | 2020-08-21 | 2023-09-05 | 苏州引航生物科技有限公司 | Modified 7 beta-hydroxysteroid dehydrogenase and application thereof |
| CN112029740A (en) * | 2020-09-15 | 2020-12-04 | 江西邦泰绿色生物合成生态产业园发展有限公司 | 7 beta hydroxysteroid dehydrogenase mutant and application thereof |
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| CN110776572B (en) | 2021-06-29 |
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