CN115261296A - Recombinant escherichia coli for expressing aminopeptidase M1AP and construction method - Google Patents
Recombinant escherichia coli for expressing aminopeptidase M1AP and construction method Download PDFInfo
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Abstract
The invention discloses a recombinant escherichia coli for expressing aminopeptidase M1AP and a construction method thereof. The invention clones the aminopeptidase gene M1AP, successfully constructs the M1AP recombinant expression plasmid pET-28a-M1AP-His with the C end fused with the His label, and takes escherichia coli as an expression host of the aminopeptidase M1AP. The recombinant Escherichia coli obtained by the invention detects cell disruption supernatant in the soluble expression of M1AP gene, the enzyme activity of aminopeptidase can be as high as 112.219U/mL, and the purity is high after purification. Overcomes the defect that a large amount of inclusion bodies are easy to form when the escherichia coli expresses the functional foreign protein bodies, and successfully realizes the more efficient soluble expression of the escherichia coli. The characteristics of high growth speed and short culture period of the escherichia coli are combined, and the requirement of mass production of the aminopeptidase in a short time is successfully met.
Description
Technical Field
The invention relates to a recombinant escherichia coli for expressing aminopeptidase M1AP and a construction method thereof, belonging to the fields of biological engineering technology and genetic engineering.
Background
Aminopeptidase (Aminopeptidase) is a protein hydrolase that catalyzes the hydrolysis of peptide and protein N-terminal amino acid residues. In the field of food processing, it can remove bitter peptides formed in protein hydrolysate, improve the palatability of products, increase free amino acids, increase the flavor of foods and the like. In addition, the aminopeptidase can also be used for preparing functional peptides, is widely applied to industries of medical diagnosis, protein sequencing, beauty and skin care and the like, and has high research significance and application value. With the increasing interest in the use of aminopeptidases in the food industry, the development of novel aminopeptidases with novel properties (optimum reaction pH, optimum reaction temperature, pH stability, temperature stability, substrate specificity, etc.) is becoming an area of increasing interest. Molecular modification of existing enzymes is one of the major strategies currently in search of aminopeptidases with new properties.
We have previously achieved secretory expression of the M1 family aminopeptidase M1AP from streptomyces cinnamomi TH2 (streptomyces cinnamoneus TH 2) in streptomyces lividans (Wan et al.2019), but it is cumbersome to purify; in addition, the streptomyces is complex to operate as an expression bacterium, the experimental period is long, and the production cost is relatively high, which brings inconvenience to subsequent characteristic research and molecular modification of M1AP. Compared with a streptomycete expression system, the escherichia coli is widely applied as a foreign protein expression host, has the advantages of fast cell growth, easy operation, high growth density and the like, and is suitable for large-scale production of target protein. In an escherichia coli expression system, a pET expression vector series is a vector series which is most widely applied, and has the advantages of easiness in transformation, strong transcription activity, high transcription efficiency and the like. However, in order to successfully express a functional foreign protein in E.coli, it is necessary to overcome the formation of a large amount of inclusion bodies from the foreign protein.
Disclosure of Invention
The invention aims to provide a recombinant escherichia coli for expressing aminopeptidase M1AP, wherein the recombinant escherichia coli expresses an aminopeptidase M1AP gene (LC 198775.1) with a nucleotide sequence shown as SEQ ID No.1, escherichia coli BL21 (DE 3) is taken as an expression host, pET-28a (+) is taken as an expression vector, and a His label is fused at the C end of the aminopeptidase M1AP.
The invention also aims to provide a construction method of the recombinant escherichia coli for expressing the aminopeptidase M1AP, which mainly comprises the following steps:
(1) Construction of subcloned plasmid pClone007-M1AP
Connecting the target fragment of the M1AP gene to a subcloning vector pClone007 in a gene synthesis manner to construct a subcloning plasmid pClone007-M1AP;
(2) Construction of recombinant expression plasmid pET-28a (+) -M1AP-His
The pClone007-M1AP plasmid is subjected to enzyme digestion by using Nco I and Xho I, an M1AP gene fragment is recovered by using a gel DNA micro-recovery kit, the M1AP gene fragment is connected to a pET-28a (+) vector with the same enzyme digestion site by using T4 efficient ligase, and is transferred into escherichia coli DH5 alpha, a positive transformant is selected for culture, and the plasmid is extracted;
(3) Construction of M1 AP-expressing Strain
The recombinant expression plasmid pET-28a-M1AP-His is introduced into escherichia coli BL21 (DE 3) through an electrical transformation method, an LB plate containing Kan (50 ug/ml) resistance is coated for screening, a positive transformant is selected for culture, and a strain is preserved.
The invention has the following beneficial effects:
the aminopeptidase M1AP gene is derived from streptomyces cinnamomi TH2, can hydrolyze a plurality of amino acid residues at the N end of peptides and proteins, and the optimal substrate is leucine. The amino acid sequences are different, the aminopeptidase activities are different, the activities of the aminopeptidase activities have great improvement potential, and the more the effective components are accumulated in the application, the stronger the activities are. The Escherichia coli is used as a common expression host, and has the advantages of clear genetic background, easy culture, low cost and the like.
The invention clones the aminopeptidase gene M1AP, successfully constructs the M1AP recombinant expression plasmid pET-28a-M1AP-His with the C end fused with the His label, and takes escherichia coli as an expression host of the aminopeptidase M1AP. The recombinant Escherichia coli obtained by the invention detects cell disruption supernatant in the soluble expression of M1AP gene, the enzyme activity of aminopeptidase can be as high as 112.219U/mL, and the purity is high after purification. Overcomes the defect that exogenous protein is easy to form a large amount of inclusion bodies when escherichia coli expresses a functional exogenous protein body, and successfully realizes the efficient soluble expression of the exogenous protein body in the escherichia coli. The characteristics of high growth speed and short culture period of the escherichia coli are combined, and the requirement of mass production of the aminopeptidase in a short time is successfully met. Lays a foundation for further developing a novel aminopeptidase based on M1AP, namely the implementation of modifying the optimal reaction temperature, the optimal reaction pH, the substrate specificity, the salt tolerance and the like of the M1AP.
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FIG. 1 shows the double restriction enzyme digestion verification of recombinant expression plasmid pET-28a (+) -M1AP-His.
FIG. 2. Double restriction enzyme digestion validation of pET-28a (+) -M1AP-His in the M1AP expression strain.
FIG. 3 SDS-PAGE analysis of M1AP expression at different temperatures and different times.
FIG. 4 SDS-PAGE gel electrophoretic analysis of M1AP expression at different IPTG concentrations.
FIG. 5 SDS-PAGE gel electrophoretic analysis of purified M1AP.
Detailed Description
The following examples are intended to illustrate the invention but are not intended to limit the scope of the invention.
Media used in protocol:
LB medium (g/L): tryptone 10, yeast powder 5, naCl 5, pH7.0, and adding agar 10g in the liquid culture medium to obtain the solid culture medium.
SOC Medium (g/L): tryptone 20, yeast powder 5, naCl 0.5, KCl 0.186 and pH7.0. After sterilization 10ml MgCl was added2(1M) and 20ml (1M) glucose.
TB medium (g/L): preparing solution A (glycerol 5, yeast extract 24, tryptone 20 to 900 ml) and solution B (KH)2PO4 2.34、K2HPO412.54 constant volume to 100 ml), respectively autoclaving and mixing evenly.
The components of the culture medium are purchased from Biotechnology engineering (Shanghai) GmbH except that the yeast powder is purchased from Beijing Solebao science and technology GmbH.
Example 1 construction of the subcloning plasmid pClone007-M1AP
The base sequence of M1AP gene (GenBank: LC 198775.1) is shown in SEQ ID NO.1, the total length is 1521bp, wherein 1-69bp is the base sequence for coding secretion signal peptide, in order to construct M1AP recombinant expression plasmid with C end fused with His label, the recombinant expression plasmid is prepared byBase synthesis and connection, the M1AP gene is successfully subcloned to a vector pClone007 (Beijing Ongzhike Biotechnology Co., ltd.), and the subcloned plasmid is named pClone007-M1AP. The synthetic base sequence comprises a base sequence from 70 th to 1518 th of the M1AP gene, and ccatgg is added at the front end of the base sequenceAT(the letter "lower case" indicates NcoI cleavage site, and the underline indicates 2 bases for preventing codon shift increase) ctcgag (Xho I cleavage site) was added to the end, and the total length of the synthesized base sequence was 1463bp.
Example 2 construction of recombinant expression plasmid pET-28a (+) -M1AP-His
The pClon 007-M1AP plasmid was digested with Nco I and Xho I, and the M1AP gene fragment was recovered with a gel DNA minirecovery kit (Guangzhou Meiji Biotech Co., ltd.), ligated with a pET-28a (+) vector digested with the same enzyme and treated with a dephosphorylating reagent (Alkaline Phosphatase, takara Co., ltd.) with T4-efficient ligase (Ligation her Ver.2, toyobo Co., ltd.), transferred into E.coli DH 5. Alpha., screened by coating an LB plate containing resistance to kanamycin (Kan, 50ug/ml, beijing Sorbon Tech Co., ltd.), and positive transformants were selected and cultured, and the plasmid was extracted with a plasmid extraction kit ((Guangzhou Meiji Biotech Co., ltd.). As shown in FIG. 1, the successfully ligated plasmid was digested simultaneously with Nco I and Xho I to form two bands of about 5.3kbp and about 1.5kbp in nucleic acid electrophoresis (1: recombinant expression plasmid; 2: double digested fragment). We named the successfully ligated recombinant expression plasmid pET-28a (+) -M1AP-His.
Example 3 construction of M1AP-expressing Strain
The recombinant expression plasmid pET-28a-M1AP-His is introduced into escherichia coli BL21 (DE 3) through an electrical transformation method, an LB plate containing Kan (50 ug/ml) resistance is coated for screening, transformants are selected for culturing, strains are stored, plasmids are extracted, nco I and Xho I double enzyme digestion identification is carried out, and T7 pommeter universal primers are utilized for sequencing identification of M1AP. As shown in FIG. 2, restriction analysis of the plasmid extracted from the preserved strain resulted in the formation of two bands of about 5.3kbp and about 1.5kbp by double restriction of Nco I and Xho I in nucleic acid electrophoresis (1: plasmid extraction from the preserved strain; 2: double restriction fragment).
The electrical conversion method is as follows:
1uL recombinant plasmid and 50uL Escherichia coli competent BL21 (DE 3) competent cells mixed, gently blow and mix. And then transferred to a pre-cooled electroporation cuvette. The water on the outer cup wall of the electric rotor was then wiped dry and placed into an electric rotor (Eppendorf Co.) and the mode selected was 2500V,5mS. After the electroporation, the sample in the cuvette was mixed with 950ul of SOC broth and revived in a shaker at 200rpm at 37 ℃ for 1 hour. An appropriate amount of the cells were spread on an LB plate containing Kan (50 ug/ml).
Example 4 expression of M1AP
In order to examine the effect of temperature and time on M1AP expression, we performed aminopeptidase activity assay on disrupted supernatants (S) of M1 AP-expressing cells cultured at three culture temperatures of 16 ℃,25 ℃ and 37 ℃ for different culture times. The enzyme activity is shown in table 1: overall, 16 ℃ enzyme activity was very low, 25 ℃ and 37 ℃ enzyme activity was higher, with 25 ℃ enzyme activity being the highest. In the data, the highest enzyme activity is 98.938 +/-2.18U/ml, corresponding conditions are 25 ℃ and 24 hours, the lowest enzyme activity is 0.103 +/-0.01U/ml, corresponding conditions are 16 ℃ and 72 hours, and the aminopeptidase activity is improved by nearly thousand times by changing the temperature and the culture time. As seen from the incubation time, there was a tendency for the enzyme activity to increase and then decrease at all three temperatures. In order to more intuitively reflect the details of the expression level of aminopeptidase at three temperatures, the disrupted supernatant (S) and disrupted pellet (Insoluble, I) of the expressed cells were subjected to SDS-PAGE gel electrophoresis. As shown in fig. 3: m1AP (53.73 kDa) was mainly present in the disrupted supernatant (S) at 25 ℃ whereas M1AP was mainly present in the disrupted precipitate (I) at 37 ℃ and the presence of M1AP band was not observed significantly at 16 ℃.
TABLE 1 enzymatic Activity of M1AP expression at different temperatures and times
* Data are presented as mean ± standard deviation.
Further, we investigated the relationship between IPTG concentration and aminopeptidase M1AP expression, and it can be seen from Table 2 that the final concentration of 0.4mM IPTG resulted in the highest activity of M1AP, which was 112.219. + -. 9.251U/mL, and that when IPTG was 1.0mM, the activity of M1AP was the lowest, which was 92.520. + -. 6.092U/mL, and that the variation in IPTG concentration resulted in an improvement of aminopeptidase activity by about 0.2-fold. Overall, the effect of the addition of IPTG on the enzyme activity was not as great as the effect of temperature changes on the enzyme activity. This was confirmed by SDS-PAGE gel electrophoretic analysis (FIG. 4): there was no significant difference in M1AP in the disrupted supernatant (S) of the expressed cells cultured at different IPTG concentrations.
In summary, the optimal expression conditions of M1 AP-expressing bacteria are: the enzyme activity can reach 112.219 plus or minus 9.251U/mL when the induction expression is carried out for 24h by 0.4mM IPTG at 25 ℃.
TABLE 2 enzyme Activity expressed by M1AP at different IPTG concentrations
| IPTG concentration (mM) | Enzyme Activity (U/ml) |
| 0.1 | 93.578±5.735 |
| 0.2 | 98.837±5.035 |
| 0.4 | 112.219±9.251 |
| 0.6 | 103.163±4.566 |
| 0.8 | 107.442±5.084 |
| 1.0 | 92.520±6.092 |
* Data are presented as mean ± standard deviation.
(1) Culture of M1 AP-expressing Strain
After the preserved M1AP expression strain is recovered, adding the recovered strain into a Kan (50 ug/ml) LB liquid culture medium, shaking at 37 ℃ for 16h on a 200rpm shaking table, adding 1.5ml of seed fermentation liquor into 50ml of Kan (50 ug/ml) TB culture medium, continuing shaking table culture, and searching temperature-time and IPTG concentration when the absorbance OD600 value detected by a spectrophotometer is about 1.5.
Temperature-time exploration: adding IPTG (final concentration is 1.0 mM), respectively carrying out shake fermentation culture at 16 ℃,25 ℃ and 37 ℃, then respectively recovering about 50ml of bacterial liquid at 8h, 16h, 24h, 48h and 72h, centrifuging at 4 ℃ for 10 minutes at 7000g, separating the supernatant from the thalli, adding 10ml of broken lysate into the thalli, carrying out crushing at the lower temperature by using an ultrasonic crusher (Ningbo Xinzhi biological science and technology Co., ltd) (power is 150W, total working time is 40min; ultrasonic on time is 4.0S; ultrasonic off time is 3.0S), centrifuging at 4 ℃ for 10 minutes to separate broken supernatant (S) and broken precipitate (I), adding 5ml of pure water into the broken precipitate to dissolve, and taking a proper amount of broken precipitate to dilute four times for later use.
IPTG concentration exploration: IPTG (final concentrations 0.1mM, 0.2mM, 0.4mM, 0.6mM, 0.8mM, 1.0mM, respectively) was added thereto, and the mixture was subjected to shake fermentation at 25 ℃ for 24 hours. The subsequent operation was the same as the temperature-time exploration.
(2) SDS-PAGE protein electrophoresis sample preparation
Disruption supernatant (S): after mixing 30uL of sample +10uL of 4 xSDS stain, it was microcentrifuged, heated at 100 ℃ for 5 minutes, and 10uL of the resulting mixture was added to the sample wells.
Disruption of the precipitate (I): after mixing the 30uL quadruplely diluted sample with 10uL 4 XSDS stain, it was subjected to microcentrifugation, heated at 100 ℃ for 5 minutes, and 10uL was added to the sample well.
(3) Enzyme Activity assay
The substrate reaction system comprises: 72ul RO water, 30ul 0.2M phosphate buffer (pH = 7.0), 6ul 50mM L-Leu-pNA (Shanghai Bigdi pharmaceutical science and technology Co., ltd.) and 12ul disruption supernatant (S). The crushed supernatant and other reactants are separately put into a PCR instrument to be preheated for 5min at 40 ℃ together, then mixed, reacted for 30min at 40 ℃ and inactivated aminopeptidase in the supernatant at 5min at 95 ℃, the mixture is moved into an enzyme label plate after being cooled to 4 ℃, and the absorbance is measured at the wavelength of 405nm (the average value is obtained by measuring three groups of each sample).
Aminopeptidase activity unit: under the condition of 40 ℃, the enzyme amount required for decomposing L-leucine-p-nitroaniline (L-Leu-pNA) for 1min to generate 1umol of p-nitroaniline is one enzyme activity unit, namely 1U.
Example 5 purification of M1AP and determination of kinetic parameters
M1AP was isolated and purified from the supernatant of disrupted cells expressed 24h at 25 ℃ with 0.4mM IPTG induction. SDS-PAGE gel electrophoresis analysis of purified M1AP is shown in FIG. 5 (1: protein band in cell disruption supernatant; 2: protein band after purification of cell disruption supernatant). The kinetic parameters of purified M1AP on L-Leu-pNA were as follows: vmax(umol·min-1·mg-1)＝89.422±4.325,Km(mM)＝1.795±0.293，Kcat(S-1)＝80.077±3.873,Kcat/Km(mmol-1·S-1)＝45.134±4.779。
(1) Purification of M1AP
And (4) purifying the crushed thallus supernatant by using a Biyunshi His tag protein purification kit. Pre-balancing 1ml of His-tag purification Resin, adding 4ml of crushed supernatant, softly and uniformly mixing, placing on a rotator, rotating at a low speed of 4 ℃ for one hour, and fully combining the recombinant protein in the supernatant with the purification Resin to obtain a sample to be purified. Passing the sample through the column directly once to obtain flow-through solution, passing through the column with 1ml of non-denaturing washing solution (50mM pH =8.0Tris-HCl, 5mM imidazole, 300mM NaCl), placing on a rotator, rotating at 4 deg.C for 2min, and repeating the operation 10 times; the column was loaded with 0.5ml of a non-denaturing eluent (50mM pH =8.0Tris-HCl,100mM imidazole, 300mM NaCl), the eluent was directly eluted, the operation was repeated 6 times, and the last three times were combined and dialyzed (0.01M pH =8.0 Tris-HCl) 2 times. The dialyzed samples were used for subsequent SDS-PAGE gel electrophoresis and kinetic parameter analysis.
(2) Determination of kinetic parameters
Preparing a reaction system (50mM pH =6.3 phosphate buffer solution) of L-Leu-pNA with a final concentration of 0.3-9.0mM, setting a kinetic cycle for 20 times and 1 min/time by using an enzyme labeling instrument (ThermoFisher company), measuring OD405 of aminopeptidase M1AP under L-Leu-pNA substrates with various concentrations at 40 ℃, calculating the enzyme activity of the aminopeptidase M1AP at the 20min, plotting by using a penultimate method, and calculating the corresponding Mie constant KmMaximum reaction velocity VmaxAnd the catalytic rate constant Kcat。
Sequence listing
<110> university of southwest
<120> recombinant escherichia coli for expressing aminopeptidase M1AP and construction method
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1521
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
atgcgtcaca gactcatcgt tccgggcgcg gccgccgtct gcctgctgct ggcgattccc 60
gcgtcggccg ccgagcccac gccgggtgcc ccgggcgtcg gggactccta ctaccccgac 120
tccgggaacg gcggatacca cgtctcccac tacgacctga ggctgaagta ccagccgaag 180
acggacaaac tggagggcac ggcgaccctg ctcgccacca ccacccagga cctgtcccgc 240
ttcaacctgg acttcctgct cgacgtcaac gaggtcctgg tcaacggcaa gaaggccacg 300
ttcgccaagt cgggcgtcca cgagctggag atcaccccgt ccgccgccct ccccaagggc 360
aaggacatca gcgtggtcgt ccgctacagc ggcgccccct cccaggtgaa ggtcaacaac 420
ttcaccgcct ggtaccgcac cccggacggc gcggtggtgg cgcaggagcc ggaggccgcc 480
gcctggtggt tccccagcaa cgaccacccg acggacaagg ccacgttcga catctcggtg 540
tcggtgccgg acggatacca gacggtcagc aacggcatcc tcggctcaca gagctccaag 600
ctcggctgga cgcgctacaa ctggcgctcc gccaagccgc aggcgacgta cctcaccacg 660
ctcgcggtgg gcaagttcga catcacgcag gacaccaccg ccaacgggct gcccgtgctc 720
aacgcctaca gcaaggacct cggcgacaac gccccggccg tgcgggcgag cgtcgagcgc 780
accggcgaga tcatcgactg ggagagctcg ctcttcgggc cgtacccctt caccgcggtc 840
ggcggctatg tgcccaacgt caagacgggc tacgcgctgg agacacagac ccggccgtac 900
tacagcccct ccagcttcgc caacggcgcc aacacctcgc tgatcgtgca cgagctcgcc 960
caccagtggt tcggcgacag cgtctcggtg aaggactgga agaacatctg gatcaacgag 1020
ggcttcgcca cctacgccca gtggctgtgg tcggagaagg agggcgaggg caccacccag 1080
gaactcgccg actacatcta caacggggtg cccgccgaca acgccttctg gacggtgaag 1140
ccgggcgacc cgggcgcgga caagcagttc gaccgcgccg tctacgaccg gggcgccctc 1200
gcgctccagg cactgcgcac caaggtcggg gacaagacgt tcttcgagat cctccagggg 1260
tggacggccg cgaacaagta cggcaacgcc tccgtgcagg acttcgcgac gttcgccgag 1320
aaggtctccg gcaagccgct ggcgcagctc ttcgacacct ggctgttcgc cacgtcgaag 1380
ccggccgtgt cgccgaacgc cacggccacg gcgggcaagg cggggaaggc ggggaaggcg 1440
gccaagtcgc tcaaggcccc ggcccagccc aagtcctgga agaagatcca ggaggcgcag 1500
gcagcccacc cgcaccactg a 1521
Claims (3)
1. A recombinant escherichia coli expressing aminopeptidase M1AP, comprising: the recombinant escherichia coli expresses an aminopeptidase M1AP gene (LC 198775.1) with a nucleotide sequence shown as SEQ ID No.1, escherichia coli BL21 (DE 3) is taken as an expression host, pET-28a (+) is taken as an expression vector, and the His label is fused at the C end of the aminopeptidase M1AP.
2. A method for constructing the recombinant E.coli expressing aminopeptidase M1AP according to claim 1, wherein: the method comprises the following steps:
(1) Construction of subcloned plasmid pClone007-M1AP
Connecting the M1AP gene target fragment to a subcloning vector pClone007 in a gene synthesis manner to construct a subcloning plasmid pClone007-M1AP;
(2) Construction of recombinant expression plasmid pET-28a (+) -M1AP-His
The pClone007-M1AP plasmid is subjected to enzyme digestion by using Nco I and Xho I, an M1AP gene fragment is recovered by using a gel DNA micro-recovery kit, the M1AP gene fragment is connected to a pET-28a (+) vector with the same enzyme digestion site by using T4 efficient ligase, and is transferred into escherichia coli DH5 alpha, a positive transformant is selected for culture, and the plasmid is extracted;
(3) Construction of M1 AP-expressing Strain
The recombinant expression plasmid pET-28a-M1AP-His is introduced into escherichia coli BL21 (DE 3) through an electrical transformation method, an LB plate containing Kan (50 ug/ml) resistance is coated for screening, a positive transformant is selected for culture, and a strain is preserved.
3. The method for efficiently expressing aminopeptidase M1AP by using the recombinant Escherichia coli of claim 1, wherein: the recombinant Escherichia coli is preferably induced to express for 24h at 25 ℃ by 0.4mM IPTG when TB medium is used.
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