CN114774380A - Expression method of high-activity hydroxysteroid dehydrogenase - Google Patents

Expression method of high-activity hydroxysteroid dehydrogenase Download PDF

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CN114774380A
CN114774380A CN202210465910.1A CN202210465910A CN114774380A CN 114774380 A CN114774380 A CN 114774380A CN 202210465910 A CN202210465910 A CN 202210465910A CN 114774380 A CN114774380 A CN 114774380A
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宋建芳
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Abstract

The invention discloses an expression method of high-activity hydroxysteroid dehydrogenase, belonging to the technical field of biological enzyme. The expression method comprises the following steps: constructing a recombinant expression plasmid containing the hydroxysteroid dehydrogenase gene by taking a plasmid pET32a as a starting vector, introducing the constructed recombinant expression plasmid into host cells, and culturing the host cells to express the hydroxysteroid dehydrogenase gene, wherein the host cells are Origami2(DE 3). The invention takes pET32a as an expression vector of 7 alpha-HSDH and 7 beta-HSDH and Origami2(DE3) as a host bacterium, and can effectively improve the expression quantity and the activity of hydroxysteroid dehydrogenase such as 7 alpha-HSDH, 7 beta-HSDH and the like.

Description

Expression method of high-activity hydroxysteroid dehydrogenase
Technical Field
The invention relates to the technical field of biological enzymes, in particular to an expression method of high-activity hydroxysteroid dehydrogenase.
Background
The fel Ursi powder is dried product of bile of animals belonging to the family Ursidae, and has effects of clearing away heat and toxic materials, suppressing hyperactive liver, improving eyesight, killing parasite, and stopping bleeding. The natural bear gall powder is characterized in that the active ingredient is tauroursodeoxycholic acid (TUDCA) and also contains taurochenodeoxycholic acid (TCDCA), and the weight ratio of TUDCA: TCDCA is between 1.0:1.0 and 1.5: 1.0. The difference of the chemical components of the poultry bile and the bear bile is as follows: taurochenodeoxycholic acid (TCDCA) is the major component in poultry bile and does not contain TUDCA. Studies have shown that TUDCA can be generated from TCDCA via a redox chemical reaction: TCDCA is catalytically converted to TUDCA by 7 α -hydroxysteroid dehydrogenase (7 α -HSDH) and 7 β -hydroxysteroid dehydrogenase (7 β -HSDH) in this order. The biological enzyme technology is applied to the oriented biosynthesis of the tauroursodeoxycholic acid compound, can convert low-value poultry bile juice products into high-added-value products, can be used as a substitute resource of the bear gall powder, can solve the problem of the source of the medicinal bear gall powder to a great extent, can also be used as a new medicinal resource, and has strong competitiveness and potential.
In the prior art, for example, Chinese patent CN107058250B is to construct 7 beta-HSDH gene on pGEX-6p-1 plasmid by gene construction, and then transfer the constructed expression vector into BL21(DE3) host bacteria by an electrotransformation or chemical transformation method, culture and express 7 alpha-HSDH or 7 beta-HSDH, and use the expression vector to directionally convert TCDCA in poultry bile into TUDCA, but the 7 alpha-HSDH and 7 beta-HSDH expressed by the method have low enzyme activity and cannot well meet the actual requirement. Chinese patent CN112852652A constructs the gene of 7 alpha-HSDH or 7 beta-HSDH into a yeast expression system, and improves the enzyme activity by the modification of the yeast expression system. However, construction of yeast is much more difficult than that of E.coli, the culture period is longer, and the expression level of enzyme is much lower than that of E.coli.
Therefore, it is necessary to provide an expression method of hydroxysteroid dehydrogenase which has simple construction of expression system and can significantly improve enzyme activity.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide an expression method of high-activity hydroxysteroid dehydrogenase, which can effectively improve the expression quantity and the enzyme activity of the hydroxysteroid dehydrogenase.
In order to solve the problems, the technical scheme adopted by the invention is as follows:
a method for expressing high-activity hydroxysteroid dehydrogenase comprises the following steps: constructing a recombinant expression plasmid containing the hydroxysteroid dehydrogenase gene, introducing the recombinant expression plasmid into a host cell, and culturing the host cell to express the hydroxysteroid dehydrogenase gene, wherein the host cell is Origami2(DE 3).
In a preferred embodiment of the present invention, pET32a is a starting vector for constructing a recombinant expression plasmid containing a hydroxysteroid dehydrogenase gene.
In a preferred embodiment of the invention, the hydroxysteroid dehydrogenase is 7 α -HSDH or 7 β -HSDH.
As a preferred embodiment of the invention, the recombinant expression plasmid is pET32a-N _ His-7 alpha-HSDH or pET32a-N _ His-7 beta-HSDH.
As a preferred embodiment of the present invention, the specific steps for introducing the recombinant expression plasmid into the host cell are as follows: the recombinant expression plasmid is added into a competent host cell Origami2(DE3), flicked and mixed evenly, placed on ice for 30 minutes, thermally shocked for 90 seconds at 42 ℃, added with SOC culture medium, subjected to shaking culture at 150rpm and 37 ℃ for 1.5 hours, uniformly coated on an ampicillin resistant plate, and placed and cultured overnight at 37 ℃ in an incubator.
As a preferred embodiment of the present invention, the introduction of IPTG as an inducer during the cultivation of the host cells induces the expression of the hydroxysteroid dehydrogenase gene.
Compared with the prior art, the invention has the beneficial effects that:
the invention takes pET32a as an expression vector of 7 alpha-HSDH and 7 beta-HSDH and Origami2(DE3) as a host bacterium, wherein the pET32a vector has a T7 transcription and translation system, and is expressed after being induced by host T7RNA polymerase. The T7RNA polymerase has an ultra-strong and specific function of starting the gene expression of a T7 promoter, when the T7RNA polymerase is completely induced, the expression of a host can be almost completely converted into the expression of a target gene, the final target gene expression product is close to the highest level after the induction for several hours, and meanwhile, the pET32a vector has a Trx protein label and can help the expressed protein to be correctly folded. Origami2(DE3) host bacteria were derived from K-12 cells and contained mutations in both thioredoxin reductase (trxB) and glutamthione reductase (gor) genes, which allowed the strain to more efficiently produce disulfide bonds in the cytoplasm and favour the formation of active disulfide-containing proteins, DE3 being a lysogenic lambda DE3 and therefore carrying a chromosomal copy of T7RNA polymerase under the lacUV5 promoter.
The 7 alpha-HSDH protein and the 7 beta-HSDH protein can form disulfide bonds in the expression and folding processes, and the correct folding of the secondary structure of the protein, particularly the correct formation of the disulfide bonds, has important influence on the activity of the protein, so that pET32a is used as an expression vector of the 7 alpha-HSDH, Origami2(DE3) is used as a host bacterium, and the expression quantity and the activity of hydroxysteroid dehydrogenases such as the 7 alpha-HSDH, the 7 beta-HSDH and the like can be effectively improved.
Detailed Description
The present invention will be described in further detail with reference to specific embodiments.
Example 1 expression method of 7 α -HSDH (7 α hydroxysteroid dehydrogenase)
A method for expressing 7 α -HSDH comprising the steps of:
s1 construction of recombinant expression plasmid containing hydroxysteroid dehydrogenase Gene
The gene sequence of 7 alpha-HSDH is shown in SEQ ID NO 1, is derived from Chinese patent CN106701707B, the target gene expression vector is pET32a, and a general biology (Anhui) corporation is entrusted to synthesize and obtain a recombinant expression plasmid; the competent host cell was Origami2(DE3) from Ubao organisms.
S2 introduction of recombinant expression plasmid into host cell
Taking out the recombinant expression plasmid pET32a-N _ His-7 alpha-HSDH from a refrigerator at the temperature of-20 ℃, thawing, taking one (100ul) of Origami2(DE3) competent cells, putting the one (100ul) on ice for thawing for 2 minutes, adding 2ul of the recombinant expression plasmid pET32a-N _ His-7 alpha-HSDH, flicking and uniformly mixing, placing the mixture on ice for 30 minutes, thermally shocking the mixture for 90 seconds at the temperature of 42 ℃, standing the mixture on ice for 2 minutes, adding 900ul of SOC culture medium, carrying out shaking culture at the temperature of 150rpm and 37 ℃ for 1.5 hours, uniformly coating 100ul of the mixture on an ampicillin resistant plate (the working concentration of ampicillin is 100ug/ml, the same below), and standing and culturing the mixture in an incubator at the temperature of 37 ℃ overnight.
S3, inducing 7 alpha-HSDH gene expression
Taking 5 test tubes, respectively adding 5ml of LB culture medium containing ampicillin, selecting 5 colonies with regular edges and moderate size, respectively inoculating the colonies in the 5 test tubes, culturing at 37 ℃ for 4 hours at 180rpm, taking 800ul as a strain, adding 2ul of IPTG (isopropyl-beta-D-thiogalactoside) into the rest culture medium, continuously culturing for 2 hours, detecting the expression quantity by SDS-PAGE, and selecting one strain with the best expression for subsequent expression.
Preparation of S4, 7 alpha-HSDH
Taking 20ul of strain, inoculating in 200ml LB culture medium containing ampicillin, shaking culturing at 37 deg.C and 180rpm for 6 hr, cooling to 20 deg.C, adding 200ul of IPTG, continuing culturing for 16 hr, centrifuging at 4000rpm, and collecting thallus. Resuspending the cells in cell disruption solution (50mM HEPES, 200mM NaCl, pH 8.0), disrupting the cells with a sonicator, centrifuging at 12000rpm for 1 hour in an ultracentrifuge, collecting the supernatant, and purifying with a HisHP column to obtain 7 α -HSDH enzyme, labeled as example 1-1; repeating the steps of S1-S4 twice, marking the obtained purified enzyme as an example 1-2 and an example 1-3, and detecting the enzyme activity.
S5, enzyme activity detection
Adding 2930ul of 100mM PB (phosphate buffer, pH 7.0) into a cuvette, adding 30ul of 50mM NADP, adding 30ul of 50mM TCDCA, uniformly mixing to serve as a blank control, returning to zero at 333nm wavelength in a spectrophotometer, adding 10ul of a diluent of purified protein solution (diluting according to actual conditions to ensure that the final absorbance of A333 is between 0.2 and 0.6), immediately mixing uniformly, reading the absorbance value after 1 minute, and calculating the enzyme activity according to the following formula:
Figure BDA0003624098900000051
wherein Vt is a total reaction volume (3 ml); df is the dilution factor; 5.8222 is extinction coefficient; 1.0 is the measuring optical path; vs is the volume of enzyme solution (0.01 ml).
Comparative example 1:
a method for expressing 7 alpha-HSDH, comprising the following steps:
the gene sequences of S1 and 7 alpha-HSDH are shown in SEQ ID NO 1 and are derived from Chinese patent CN106701707B, the target gene expression vector is pGEX-6p-1, and the recombinant expression plasmid is obtained by entrusting the synthesis of general biology (Anhui) corporation; the competent host cell was Origami2(DE3) from Ubao organisms.
S2 introduction of recombinant expression plasmid into host cell
Taking out the recombinant expression plasmid pGEX-6p-1-N _ GST-7 alpha-HSDH from a refrigerator at the temperature of-20 ℃, thawing, taking one competent cell (100ul) of Origami2(DE3), thawing for 2 minutes on ice, adding 2ul of the recombinant expression plasmid pET32a-N _ His-7 alpha-HSDH, flicking and uniformly mixing, placing on ice for 30 minutes, thermally shocking at 42 ℃ for 90 seconds, standing on ice for 2 minutes, adding 900ul of SOC culture medium, shaking and culturing at the temperature of 150rpm and 37 ℃ for 1.5 hours, taking 100ul of the recombinant expression plasmid pET32a-N _ His-7 alpha-HSDH, uniformly spreading on an ampicillin resistant plate, sealing, inverting in an incubator, standing at the temperature of 37 ℃ and culturing overnight.
S3, induction of 7 alpha-HSDH gene expression
Taking 5 test tubes, respectively adding 5ml of LB culture medium containing ampicillin, selecting 5 colonies with regular edges and moderate size, respectively inoculating in 5 test tubes, culturing at 180rpm and 37 ℃ for 4 hours, taking 800ul as a strain, adding 2ul of IPTG (isopropyl-beta-D-thiogalactoside) into the rest culture medium, continuously culturing for 2 hours, detecting the expression level by SDS-PAGE, and selecting one strain with best expression for subsequent expression.
Preparation of S4, 7 alpha-HSDH
Taking 20ul of strain, inoculating in 200ml LB culture medium containing ampicillin, culturing at 37 deg.C under shaking at 180rpm for 6 hr, cooling to 20 deg.C, adding 200ul IPTG, culturing for 16 hr, centrifuging at 4000rpm, and collecting thallus. Resuspending the cells in cell disruption solution (50mM HEPES, 200mM NaCl, pH 8.0), disrupting the cells with an ultrasonicator, centrifuging at 12000rpm for 1 hour with an ultracentrifuge, collecting the supernatant, and purifying with a GSTHP column to obtain 7 α -HSDH enzyme, labeled as comparative example 1-1; repeating the steps of S1-S4 twice, marking the obtained purified enzyme as a comparative example 1-2 and a comparative example 1-3, and detecting the enzyme activity.
Comparative example 2:
comparative example 2 differs from comparative example 1 in that: the starting vector was pBV220, the recombinant plasmid vector obtained by construction was pBV220-N _ His-7a-HSDH, the purification column was a HisHP column, and the other operating procedures were the same as in comparative example 1. The purified enzymes obtained were labeled as comparative example 2-1, comparative example 2-2, and comparative example 2-3.
Effect example 1:
the expression level and the enzyme activity of the purified enzymes obtained in example 1 and comparative examples 1 to 2 were measured, and the results are shown in Table 1.
TABLE 1 expression level and enzyme activity results of 7 alpha-HSDH prepared in example 1 and comparative examples 1-2
Number of expression Amount of purified enzyme (mg) Enzyme activity (U/ml)
Example 1-1 15.46 245
Examples 1 to 2 19.33 227
Examples 1 to 3 15.19 248
Comparative examples 1 to 1 3.25 135
Comparative examples 1 to 2 3.41 142
Comparative examples 1 to 3 2.37 178
Comparative example 2 to 1 0.31 13
Comparative examples 2 to 2 0.12 15
Comparative examples 2 to 3 0.32 9
As shown in Table 1, pET32a expressed the enzyme of 7. alpha. -HSDH in a larger amount and with higher activity when used as an expression vector.
Comparative example 3:
a method for expressing 7 alpha-HSDH, comprising the following steps:
the gene sequences of S1 and 7 alpha-HSDH are shown in SEQ ID NO:1, and are derived from Chinese patent CN106701707B, a target gene expression vector is pET32a, and universal biology (Anhui) corporation is entrusted to synthesize and obtain recombinant expression plasmids; the competent host cell is BL21(DE3) and is derived from Youbao organisms.
S2 introduction of recombinant expression plasmid into host cell
Taking out the recombinant expression plasmid pET32a-N _ His-7 alpha-HSDH from a refrigerator at the temperature of-20 ℃, thawing, taking one (100ul) competent cell of BL21(DE3), thawing for 2 minutes on ice, adding 2ul of the recombinant expression plasmid pET32a-N _ His-7 alpha-HSDH, flicking and uniformly mixing, placing on ice for 30 minutes, thermally shocking at the temperature of 42 ℃ for 90 seconds, standing on ice for 2 minutes, adding 900ul of SOC culture medium, carrying out shake culture at the temperature of 150rpm and 37 ℃ for 1.5 hours, taking 100ul of the recombinant expression plasmid pET32, uniformly spreading on an ampicillin resistant plate, and carrying out standing culture at the temperature of 37 ℃ in an incubator overnight.
S3, induction of 7 alpha-HSDH gene expression
Taking 5 test tubes, respectively adding 5ml of LB culture medium containing ampicillin, selecting 5 colonies with regular edges and moderate size, respectively inoculating the colonies in the 5 test tubes, culturing at 37 ℃ for 4 hours at 180rpm, taking 800ul as a strain, adding 2ul of IPTG (isopropyl-beta-D-thiogalactoside) into the rest culture medium, continuously culturing for 2 hours, detecting the expression quantity by SDS-PAGE, and selecting one strain with the best expression for subsequent expression.
Preparation of S4, 7 alpha-HSDH
Taking 20ul of strain, inoculating in 200ml LB culture medium containing ampicillin, shaking culturing at 37 deg.C and 180rpm for 6 hr, cooling to 20 deg.C, adding 200ul of IPTG, continuing culturing for 16 hr, centrifuging at 4000rpm, and collecting thallus. Resuspending the cells with cell disruption solution (50mM HEPES, 200mM NaCl, pH 8.0), disrupting the cells with an ultrasonic disruptor, centrifuging for 1 hour at 20000rpm with an ultracentrifuge, collecting the supernatant, and purifying with HisHP column to obtain 7 α -HSDH enzyme, labeled as comparative example 3-1; repeating the steps of S1-S4 twice, marking the obtained purified enzymes as a comparative example 3-2 and a comparative example 3-3, and detecting the enzyme activity.
Comparative example 4:
comparative example 4 differs from comparative example 3 in that: the host cell was HB101, and the other procedures were the same as in comparative example 3. The obtained purified enzymes were designated comparative example 4-1, comparative example 4-2, and comparative example 4-3.
Effect example 2:
the expression level and the enzyme activity of the purified enzymes obtained in example 1 and comparative examples 3 to 4 were measured, and the results are shown in table 2.
TABLE 2 expression level and enzyme activity results of 7 alpha-HSDH prepared in example 1 and comparative examples 3 to 4
Number of expression Amount of purified enzyme (mg) Enzyme activity (U/ml)
Examples 1 to 1 15.46 245
Examples 1 to 2 19.33 227
Examples 1 to 3 15.19 248
Comparative example 3-1 11.08 142
Comparative examples 3 and 2 11.25 152
Comparative examples 3 to 3 14.37 139
Comparative example 4-1 0 0
Comparative examples 4 and 2 0 0
Comparative examples 4 to 3 0 0
As is clear from Table 2, the 7. alpha. -HSDH enzyme expressed by Origami2(DE3) as a host cell had a larger amount and a higher activity, and was not expressed in HB101 strain.
Example 2: expression method of 7 beta-HSDH (7 beta hydroxysteroid dehydrogenase)
A method for expressing 7 β -HSDH comprising the steps of:
s1 construction of recombinant expression plasmid containing hydroxysteroid dehydrogenase Gene
The gene sequence of 7 beta-HSDH is shown in SEQ ID NO. 2, and is derived from Chinese patent CN107058250B, the target gene expression vector is pET32a, and a general biology (Anhui) corporation is entrusted to synthesize and obtain recombinant expression plasmid; the competent host cell was Origami2(DE3) from Youbao organisms.
S2 introduction of recombinant expression plasmid into host cell
Taking out the recombinant expression plasmid pET32a-N _ His-7 beta-HSDH from a refrigerator at the temperature of-20 ℃, thawing, taking one part (100ul) of Origami2(DE3) competent cell, putting the part on ice for thawing for 2 minutes, adding 2ul of an expression vector pET32a-N _ His-7 beta-HSDH, flicking and uniformly mixing, putting the part on ice for 30 minutes, thermally shocking the part at the temperature of 42 ℃ for 90 seconds, standing the part on ice for 2 minutes, adding 900ul of SOC culture medium, carrying out shaking culture at the temperature of 150rpm and 37 ℃ for 1.5 hours, taking 100ul of the mixture, uniformly spreading the mixture on an ampicillin resistant plate, and carrying out standing culture at the temperature of 37 ℃ in an incubator overnight.
S3, inducing 7 beta-HSDH gene expression
Taking 5 test tubes, respectively adding 5ml of LB culture medium containing ampicillin, selecting 5 colonies with regular edges and moderate size, respectively inoculating in 5 test tubes, culturing at 37 ℃ for 4 hours at 180rpm, taking 800ul as a strain, adding 2ul of IPTG (isopropyl-beta-D-thiogalactoside) into the rest culture medium, continuously culturing for 2 hours, detecting the expression quantity by SDS-PAGE, and selecting one strain with the best expression for subsequent expression.
Preparation of S4, 7 beta-HSDH
Taking 20ul of strain, inoculating in 200ml LB culture medium containing ampicillin, culturing at 37 deg.C under shaking at 180rpm for 6 hr, cooling to 20 deg.C, adding 200ul IPTG, culturing for 16 hr, centrifuging at 4000rpm, and collecting thallus. Resuspending the cells in cell disruption medium (50mM HEPES, 200mM NaCl, pH 8.0), disrupting the cells with an ultrasonic disruptor, centrifuging at 12000rpm for 1 hour with an ultracentrifuge, collecting the supernatant, and purifying with a HisHP column to obtain 7 β -HSDH enzyme, labeled as example 2-1; repeating the steps of S1-S4 twice, marking the obtained purified enzyme as an example 2-2 and an example 2-3, and detecting the enzyme activity.
S5, enzyme activity detection
Adding 2930ul of 100mM PB (phosphate buffer, pH 7.0) into a cuvette, adding 30ul of 50mM NADP, adding 30ul of 50mM TUDCA, uniformly mixing to serve as a blank control, returning to zero at 333nm wavelength in a spectrophotometer, adding 10ul of a diluent of purified protein solution (diluting according to actual conditions to ensure that the final absorbance of A333 is between 0.2 and 0.6), immediately mixing uniformly, reading the absorbance value after 1 minute, and calculating the enzyme activity according to the following formula:
Figure BDA0003624098900000101
wherein Vt is a total reaction volume (3 ml); df is the dilution factor; 5.8222 is extinction coefficient; 1.0 is a measuring optical path; vs is the volume of enzyme solution (0.01 ml).
Comparative example 5:
a method for expressing 7 β -HSDH comprising the steps of:
the gene sequences of S1 and 7 beta-HSDH are shown in SEQ ID NO:2, the target gene expression vector is pGEX-6p-1, and a recombinant expression plasmid is obtained by entrusting the synthesis of a general biology (Anhui) corporation; the competent host cell was Origami2(DE3) from Youbao organisms.
S2 introduction of recombinant expression plasmid into host cell
Taking out the recombinant expression plasmid pGEX-6p-1-N _ GST-7 beta-HSDH from a refrigerator at the temperature of-20 ℃, thawing, taking one competent cell (100ul) of Origami2(DE3), putting on ice for thawing for 2 minutes, adding 2ul of the recombinant expression plasmid pET32a-N _ His-7 beta-HSDH, flicking and uniformly mixing, placing on ice for 30 minutes, thermally shocking at 42 ℃ for 90 seconds, standing on ice for 2 minutes, adding 900ul of SOC culture medium, carrying out shaking culture at the temperature of 150rpm and 37 ℃ for 1.5 hours, taking 100ul of the recombinant expression plasmid, uniformly coating on an ampicillin resistant plate, sealing, inverting in an incubator, standing at the temperature of 37 ℃ overnight for culture.
S3, inducing 7 beta-HSDH gene expression
Taking 5 test tubes, respectively adding 5ml of LB culture medium containing ampicillin, selecting 5 colonies with regular edges and moderate size, respectively inoculating in 5 test tubes, culturing at 180rpm and 37 ℃ for 4 hours, taking 800ul as a strain, adding 2ul of IPTG (isopropyl-beta-D-thiogalactoside) into the rest culture medium, continuously culturing for 2 hours, detecting the expression level by SDS-PAGE, and selecting one strain with best expression for subsequent expression.
Preparation of S4, 7 beta-HSDH
Taking 20ul of strain, inoculating in 200ml LB culture medium containing ampicillin, culturing at 37 deg.C under shaking at 180rpm for 6 hr, cooling to 20 deg.C, adding 200ul IPTG, culturing for 16 hr, centrifuging at 4000rpm, and collecting thallus. Resuspending the cells with cell disruption solution (50mM HEPES, 200mM NaCl, pH 8.0), disrupting the cells with an ultrasonic disruptor, centrifuging at 12000rpm for 1 hr with an ultracentrifuge, collecting the supernatant, and purifying with a GSTHP column to obtain 7 β -HSDH enzyme, labeled as comparative example 5-1; repeating the steps of S1-S4 twice, marking the obtained purified enzymes as a comparative example 5-2 and a comparative example 5-3, and detecting the enzyme activity.
Comparative example 6:
comparative example 6 differs from comparative example 5 in that: the starting vector was pBV220, the recombinant plasmid vector obtained by construction was pBV220-N _ His-7. beta. -HSDH, the purification column was a HisHP column, and the other operation procedures were the same as in comparative example 5. The purified enzymes obtained were labeled as comparative example 6-1, comparative example 6-2, and comparative example 6-3.
Effect example 3:
the expression level and the enzyme activity of the purified enzymes obtained in example 2 and comparative examples 5 to 6 were measured, and the results are shown in Table 3.
TABLE 3 expression level and enzyme activity results of 7 beta-HSDH prepared in example 2 and comparative examples 5 to 6
Number of expression Amount of purified enzyme (mg) Enzyme activity (U/ml)
Example 2-1 19.58 133
Examples 2 to 2 17.36 129
Examples 2 to 3 17.63 135
Comparative example 5-1 3.80 41
Comparative examples 5 and 2 5.14 37
Comparative examples 5 to 3 3.28 52
Comparative example 6-1 0.42 2
Comparative examples 6 to 2 0.27 7
Comparative examples 6 to 3 0.23 9
As shown in Table 3, pET32a expressed more amount of 7. beta. -HSDH enzyme and had higher activity when used as an expression vector.
Comparative example 7:
a method for expressing 7 beta-HSDH, comprising the following steps:
the gene sequences of S1 and 7 beta-HSDH are shown in SEQ ID NO:2, the target gene expression vector is pET32a, and a general purpose organism (Anhui) corporation is entrusted to synthesize and obtain recombinant expression plasmid; the competent host cell is BL21(DE3) and is derived from Youbao organisms.
S2 introduction of recombinant expression plasmid into host cell
Taking out the recombinant expression plasmid pET32a-N _ His-7 beta-HSDH from a refrigerator at the temperature of-20 ℃, thawing, taking one (100ul) competent cell of BL21(DE3), thawing for 2 minutes on ice, adding 2ul expression vector pET32a-N _ His-7 beta-HSDH, flicking and uniformly mixing, placing on ice for 30 minutes, thermally shocking at the temperature of 42 ℃ for 90 seconds, standing on ice for 2 minutes, adding 900ul SOC culture medium, carrying out shake culture at the temperature of 150rpm and 37 ℃ for 1.5 hours, taking 100ul uniformly and coating on an ampicillin resistant plate, and carrying out standing culture at the temperature of 37 ℃ in an incubator overnight.
S3, induction of 7 beta-HSDH gene expression
Taking 5 test tubes, respectively adding 5ml of LB culture medium containing ampicillin, selecting 5 colonies with regular edges and moderate size, respectively inoculating the colonies in the 5 test tubes, culturing at 37 ℃ for 4 hours at 180rpm, taking 800ul as a strain, adding 2ul of IPTG (isopropyl-beta-D-thiogalactoside) into the rest culture medium, continuously culturing for 2 hours, detecting the expression quantity by SDS-PAGE, and selecting one strain with the best expression for subsequent expression.
Preparation of S4, 7 beta-HSDH
Taking 20ul of strain, inoculating in 200ml LB culture medium containing ampicillin, culturing at 37 deg.C under shaking at 180rpm for 6 hr, cooling to 20 deg.C, adding 200ul IPTG, culturing for 16 hr, centrifuging at 4000rpm, and collecting thallus. Resuspending the cells in cell disruption solution (50mM HEPES, 200mM NaCl, pH 8.0), disrupting the cells with an ultrasonicator, centrifuging at 20000rpm for 1 hour with an ultracentrifuge, collecting the supernatant, and purifying with HisHP column to obtain 7 β -HSDH enzyme, labeled as comparative example 7-1; repeating the steps of S1-S4 twice, marking the obtained purified enzymes as a comparative example 7-2 and a comparative example 7-3, and detecting the enzyme activity.
Comparative example 8:
comparative example 8 differs from comparative example 7 in that: the host cell was HB101, and the other procedures were the same as in comparative example 3. The obtained purified enzymes were labeled comparative example 8-1, comparative example 8-2, and comparative example 8-3.
Effect example 4:
the expression level and the enzyme activity of the purified enzymes obtained in example 2 and comparative examples 7 to 8 were measured, and the results are shown in table 4.
TABLE 4 expression level and enzyme activity results of 7 beta-HSDH prepared in example 2 and comparative examples 7-8
Number of expression Amount of purified enzyme (mg) Enzyme activity (U/ml)
Example 2-1 19.58 133
Examples 2 to 2 17.36 129
Examples 2 to 3 17.63 135
Comparative example 7-1 11.66 52
Comparative example 7-2 10.34 45
Comparative examples 7 to 3 13.78 41
Comparative example 8-1 0 0
Comparative examples 8 and 2 0 0
Comparative examples 8 to 3 0 0
As is clear from Table 4, the 7. beta. -HSDH enzyme expressed by Origami2(DE3) as a host cell had a larger amount and a higher activity, and was not expressed in HB101 strain.
The above embodiments are only preferred embodiments of the present invention, and the scope of the present invention should not be limited thereby, and any insubstantial changes and substitutions made by those skilled in the art based on the present invention are intended to be covered by the claims.
SEQUENCE LISTING
<110> Song Jian Fang
<120> expression method of high-activity hydroxysteroid dehydrogenase
<130> 20220420
<160> 2
<170> PatentIn version 3.3
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<211> 804
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<213> Artificial Synthesis
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gaattagcca aggaagttat atccaacatt gaaagtaatg gtggtagagc taagttcgta 180
tatttcaatg ctcgtgagcc acaaacctat actactatgg tagaaactgt ggcacaaaat 240
gaaggaaggt tagacatatt agtaaataac tacggtgaaa ctaacgtaaa gctcgacaga 300
gatttagtta atggggacac agaggaattt tttaggatag ttcaagataa cttacaaagc 360
gtttatttac ctagtaaggc tgcaatacct cgtatggcta aaaatggagg tggaagtata 420
gtaaatatat caacaatagg atctgttgtt ccagacttag gaaggattgc ttattgtgtt 480
tcaaaggcag caataaactc tttaactcaa aatatagctc ttcaatatgc gagacaaggg 540
gtaagatgta atgctgtgct tccaggctta attggaacta aagcagctat ggagaatatg 600
accgatgaat ttagggattc cttcttaaga catgtaccaa taaacagagt cggaaaacca 660
gaagatattg caaaggcagt actttattat gcaagtgatg attcagatta tgtaactgga 720
atgattcatg aagttgctgg aggatatgct ttaggaagtc cacaatatgc tgagttttct 780
gcaatgatgg agagaagtag atag 804
<210> 2
<211> 798
<212> DNA
<213> Artificial Synthesis
<400> 2
atgaatatga atttaagaga aaaatatgga gaatggggaa ttatattagg tgctactgaa 60
ggtgtaggaa aagccttttg tgaaaaaatc gctgctggtg gaatgaatgt agtaatggta 120
ggcagaagag aggagatgtt aaaggactta ggtagagaaa taagtaataa atatggagtt 180
gaacatttag taataaaggc agattttgca gatccatcat ctgtggacaa gatatttgag 240
caaactaagg aattagatat gggattcatg tcttatgttg cttgcttcca tacatttggt 300
aagttacaag atacgccttg ggaaaaacat gagcaaatga taaatgtaaa tgttattaca 360
ttttttaaat gtttttatca ttatatgggt atatttgcaa agcaagatag aggggctatt 420
ataaatgtat catctcttac tggaataagt agttcacctt ataatgctca atatggtgca 480
ggaaaatcat atatattaaa gttaacagaa gcagttgctt gtgaagctgc taagacaaat 540
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cctggtggac cagctggaga agcagtaatg aaatcagcat taactccaga ggcatgtgtt 660
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aaaaatgtac acaactggaa agctaatcat acagctgatg agtatataac atatatgggg 780
tctttctatg agaaataa 798

Claims (6)

1. A method for expressing high-activity hydroxysteroid dehydrogenase comprises the following steps: constructing a recombinant expression plasmid containing a hydroxysteroid dehydrogenase gene, introducing the constructed recombinant expression plasmid into a host cell, and culturing the host cell to express the hydroxysteroid dehydrogenase gene, wherein the recombinant expression plasmid comprises: the host cell is Origami2(DE 3).
2. The method for expressing hydroxysteroid dehydrogenase as set forth in claim 1, wherein: the starting vector for constructing the recombinant expression plasmid containing the hydroxysteroid dehydrogenase gene was pET32 a.
3. The method for expressing hydroxysteroid dehydrogenase as set forth in claim 1 or 2, wherein: the hydroxysteroid dehydrogenase is 7 alpha-HSDH or 7 beta-HSDH.
4. The method for expressing hydroxysteroid dehydrogenase as set forth in claim 1, wherein: the recombinant expression plasmid is pET32a-N _ His-7 alpha-HSDH or pET32a-N _ His-7 beta-HSDH.
5. The method for expressing a high-activity hydroxysteroid dehydrogenase as claimed in claim 1, wherein: the specific steps for introducing the recombinant expression plasmid into a host cell are as follows: the recombinant expression plasmid was added to competent host cell Origami2(DE3), gently mixed, placed on ice for 30 minutes, heat-shocked at 42 ℃ for 90 seconds, added to SOC culture medium, subjected to shaking culture at 150rpm at 37 ℃ for 1.5 hours, then uniformly spread on an ampicillin resistant plate, and subjected to overnight standing culture at 37 ℃ in an incubator.
6. The method for expressing a high-activity hydroxysteroid dehydrogenase as claimed in claim 1, wherein: adding inducer IPTG to induce the expression of hydroxyl steroid dehydrogenase gene during the process of culturing host cells.
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114250205A (en) * 2021-12-28 2022-03-29 宋建芳 7 alpha-hydroxysteroid dehydrogenase mutant with high thermal stability and application thereof

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106635941A (en) * 2016-11-07 2017-05-10 江南大学 Thermophilic esterase derived from aquifex aeolicus strain and functional verification of thermophilic esterase

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106635941A (en) * 2016-11-07 2017-05-10 江南大学 Thermophilic esterase derived from aquifex aeolicus strain and functional verification of thermophilic esterase

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114250205A (en) * 2021-12-28 2022-03-29 宋建芳 7 alpha-hydroxysteroid dehydrogenase mutant with high thermal stability and application thereof
CN114250205B (en) * 2021-12-28 2022-11-11 宋建芳 7 alpha-hydroxysteroid dehydrogenase mutant with high thermal stability and application thereof

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