CN105950580A - Preparation method of protein UGTCs4 - Google Patents
Preparation method of protein UGTCs4 Download PDFInfo
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Abstract
The invention discloses a preparation method of protein UGTCs4. The protein UGTCs4 has the croctin acid glycosyl transferase activity. The preparation method comprises the following step of culturing engineering bacteria, so as to obtain the protein UGTCs4, wherein the engineering bacteria is a recombinant bacteria which is obtained by importing a recombinant expression carrier into host bacteria, and the recombinant expression carrier is a recombinant plasmid which is obtained by inserting the expression carrier into an encoding gene of the protein UGTCs4. After proofing by experiments, the preparation method of the protein UGTCs4 has the advantages that the croctin acid can be sequentially glycosylated to form crocin 5 and crocin 3; the important application value is realized for the production of crocin 5 and/or crocin 3, preparation of products with croctin acid glycosyl transferase functions, or catalyzing on glycosylation of croctin acids.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of method preparing protein UGTCs4.
Background technology
Stigma Croci is again Stigma Croci, Stigma Croci, for perennial irides Stigma Croci (Crocus sativus L.)
Dry stigma, be a kind of rare Chinese medicine of China.Stigma Croci total glycosides is the pigment compound extracted from Stigma Croci,
It is mainly composed of crocin (crocus), crocetin (crocetin) and derivant, wherein crocin
It is a series of ester glycosides of being combined into different sugar of crocetin, is one of croceous main active, has anti-
Platelet aggregation, antithrombus formation, resist myocardial ischemia, the multiple pharmacological effect such as anticancer, and almost non-toxic side effect.
Now there are some researches show, crocetin glycosyl transferase has glycosylation crocetin and forms the function of crocin, as
Morgan in 2004 etc. separate discovery UGTCs2 gene (GeneID:AY36026), its protokaryon from Stigma Croci style
The crude protein expressed has glycosylation crocetin and forms the function of crocin, therefore by UGTCs2 gene annotation for hiding
The encoding gene of Flos Carthami acid glycosyl transferase；For another example Mai Nagatoshi in 2012 separates discovery from Fructus Gardeniae cell
UGT75L6 gene (GeneID:AB555731) and UGT94E5 gene (GeneID:555739), UGT75L6 base
Crocin glycosides 5, crocin glycosides 3 is formed because the protein crude extract administration of prokaryotic expression has order glycosylation crocetin
With the function of crocin glycosides 4, the protein crude extract administration of UGT94E5 gene prokaryotic has glycosylation crocin glycosides 5
Form crocin glycosides 2 and the function of crocin glycosides 1, therefore, by UGT75L6 gene and UGT94E5 gene also
All annotations are the encoding gene of crocetin glycosyl transferase.But so far, still cannot obtain highly purified Stigma Croci
Acid glycosyl transferase.
Summary of the invention
The technical problem to be solved is the protein how prepared and have crocetin glycosyl transferase activity.
For solving above-mentioned technical problem, present invention firstly provides a kind of method preparing protein UGTCs4, described
Protein UGTCs4 has crocetin glycosyl transferase activity.
The method preparing protein UGTCs4 provided by the present invention, it may include following steps: culturing engineering bacterium,
To described protein UGTCs4；Described engineering bacteria is that recombinant expression carrier imports the recombinant bacterium that Host Strains obtains；Institute
Stating recombinant expression carrier is that the encoding gene of described protein UGTCs4 is inserted the recombiant plasmid that expression vector obtains；
Described protein UGTCs4 can be following a1) or a2) or a3) or a4):
A1) protein shown in sequence 2 during aminoacid sequence is sequence table；
A2) in sequence table the N end of the protein shown in sequence 2 or/and C end connects the fused protein that obtains of label；
A3) protein shown in sequence 4 during aminoacid sequence is sequence table；
A4) by a1) or a2) or a3) shown in protein through one or several amino acid residue replacement and/or
The protein with crocetin glycosyl transferase activity that disappearance and/or interpolation obtain.
Wherein, in sequence table, sequence 2 can be made up of 459 amino acid residues；In sequence table, sequence 4 can be by 492
Amino acid residue forms.
In order to make a1) in protein be easy to purification, can in sequence table the amino of the protein shown in sequence 2
End or carboxyl terminal connect upper label as shown in table 1.
Table 1, the sequence of label
| Label | Residue | Sequence |
| Poly-Arg | 5-6 (usually 5) | RRRRR |
| FLAG | 8 | DYKDDDDK |
| Strep-tag II | 8 | WSHPQFEK |
| c-myc | 10 | EQKLISEEDL |
Above-mentioned a4) in protein, one or the replacement of several amino acid residue and/or disappearance and/or be added to
Less than the replacement of 10 amino acid residues and/or disappearance and/or interpolation.
Above-mentioned a4) in protein can synthetic, it is possible to first synthesize its encoding gene, then carry out biological expression and obtain.
Above-mentioned a4) in the encoding gene of protein can be by by sequence in sequence table 1 or sequence table shown in sequence 3
DNA sequence in lack the codon of one or several amino acid residue, and/or carry out the mistake of one or several base pair
Justice sudden change, and/or hold the coded sequence connecting the label shown in table 1 to obtain at its 5 ' end and/or 3 '.
In said method, the encoding gene of described protein UGTCs4 concretely following b1) or b2) or b3) or
B4) DNA molecular shown in:
B1) DNA molecular shown in sequence 1 during nucleotide sequence is sequence table；
B2) DNA molecular shown in sequence 3 during nucleotide sequence is sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and coding right is wanted
Seek the DNA molecular of protein UGTCs4 described in 1；
B4) under strict conditions with b1) or b2) institute in the nucleotide sequence hybridization that limits, and coding claim 1
State the DNA molecular of protein UGTCs4.
Wherein, described nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；Described nucleic acid divides
Son can also be RNA, such as mRNA or hnRNA etc..
Wherein, in sequence table, sequence 1 is made up of 1377 nucleotide, the nucleotide coding sequence of sequence 1 in sequence table
Aminoacid sequence shown in sequence 2 in table；In sequence table, sequence 3 is made up of 1476 nucleotide, sequence in sequence table
Aminoacid sequence shown in sequence 4 in the nucleotide coding sequence table of 3.
Those of ordinary skill in the art can use the side of known method, such as orthogenesis and point mutation easily
Method, suddenlys change to the nucleotide sequence of code for said proteins UGTCs4 of the present invention.Those are through manually modified
, there is the nucleotide sequence 75% of described protein UGTCs4 with isolated of the present invention or higher homogeneity
Nucleotide, as long as code for said proteins UGTCs4, is all derived from the nucleotide sequence of the present invention and is equal to this
The sequence of invention.
Term used herein " homogeneity " refers to the sequence similarity with native sequence nucleic acid." homogeneity " includes and this
The nucleoside of the protein of the sequence 2 of the polynucleotide of invention or the composition of the aminoacid sequence shown in sequence 4 of sequence table
Acid sequence has 75% or higher, or 80% or higher, or 85% or higher, or 90% or higher, or 95% or higher
The nucleotide sequence of homogeneity.Homogeneity can with the naked eye or computer software is evaluated.Use computer software,
Homogeneity between two or more sequences can use percentage ratio (%) to represent, it can be used to evaluate between correlated series
Homogeneity.
In said method, described recombinant expression carrier can be the sequence of the multiple clone site insertion sequence table at expression vector
The recombiant plasmid that DNA molecular shown in 1 obtains.
Described expression vector can be carrier pET-28a (+).
In said method, described recombinant expression carrier concretely carrier pET-28a (+) Nde I recognition site
And the recombiant plasmid that between Sal I recognition site, the DNA molecular shown in sequence 1 of insertion sequence table obtains
pET28a-UGTCs4.The protein shown in sequence 4 of recombiant plasmid pET28a-UGTCs4 expressed sequence table.
In said method, described Host Strains can be escherichia coli.
Described escherichia coli can be E. coli BL21 (DE3).
In said method, during described " culturing engineering bacterium ", carry out IPTG induction.
In said method, the method for described " IPTG induction " is as follows: as the OD of cultivating system600nmValue is 0.3～0.4
Time, add IPTG and make its concentration in cultivating system be 50 μMs～600 μMs (as 50 μMs, 100 μMs, 200
μM, 300 μMs, 400 μMs, 500 μMs, 600 μMs, 50 μMs～100 μMs, 50 μMs～200 μMs, 50 μMs～
300 μMs, 50 μMs～400 μMs, 50 μMs～500 μMs, 50 μMs～600 μMs, 100 μMs～200 μMs, 100 μMs～
300 μMs, 100 μMs～400 μMs, 100 μMs～500 μMs, 100 μMs～600 μMs, 200 μMs～300 μMs, 200
μM～400 μMs, 200 μMs～500 μMs, 200 μMs～600 μMs, 300 μMs～400 μMs, 300 μMs～500 μMs,
300 μMs～600 μMs, 400 μMs～500 μMs, 400 μMs～600 μMs or 500 μMs～600 μMs).
Described " add IPTG make its concentration in cultivating system be 50 μMs～600 μMs " refers to described IPTG
Initial concentration in cultivating system.
In said method, described " culturing engineering bacterium " can comprise the steps:
(1) cultivate under the conditions of 18 DEG C～20 DEG C (such as 18 DEG C or 20 DEG C), until the OD of cultivating system600nm
Value is 0.3～0.4；
(2), after completing step (1), add IPTG and to make its concentration in cultivating system be 50 μMs～600 μMs,
18 DEG C～20 DEG C (such as 18 DEG C or 20 DEG C) cultivate 6h～18h (as 6h, 8h, 10h, 12h, 14h, 16h, 18h,
6h～8h, 6h～10h, 6h～12h, 6h～14h, 6h～16h, 6h～18h, 8h～10h, 8h～12h, 8h～
14h, 8h～16h, 8h～18h, 10h～12h, 10h～14h, 10h～16h, 10h～18h, 12h～14h, 12h～
16h, 12h～18h, 14h～16h, 14h～18h or 16h～18h).
In described step (1), described cultivation can be shaken cultivation.The rotating speed of described shaken cultivation concretely 100rpm～
140rpm, can be more specifically 120rpm.
In described step (2), described cultivation can be shaken cultivation.The rotating speed of described shaken cultivation concretely 100rpm～
140rpm, can be more specifically 120rpm.
In described step (2), described " add IPTG and to make its concentration in cultivating system be 50 μMs～600 μ
M " in concentration refer to described IPTG initial concentration in cultivating system.
In said method, described " culturing engineering bacterium " may also include step (3): after completing described step (2), from
The heart collects thalline.Parameter of noncentricity can be that 5000rpm is centrifuged 10min.
In said method, described " culturing engineering bacterium " may also include step (4): takes the bacterium that described step (3) obtains
Body, crushes described thalline, and (this supernatant is containing protein UGTCs4 to be then centrifuged for collecting supernatant
Crude protein solution).Parameter of noncentricity can be 4 DEG C, 13000rpm is centrifuged 30min.
In said method, described " culturing engineering bacterium " may also include step (5): take that described step (4) obtains is upper
Clear liquid, carries out affinity chromatography purification, solution (being protein UGTCs4 solution) after obtaining post.
In described step (5), described affinity chromatography purification specifically includes first uses lysis buffer balance nickel post, so
The supernatant that step (4) described in rear loading obtains, more successively with the lavation buffer solution containing 20mmol/L imidazoles, contain
The lavation buffer solution of 80mmol/L imidazoles and the lavation buffer solution containing 100mmol/L imidazoles rinse nickel post, finally with containing
The lavation buffer solution of 250mmol/L imidazoles rinses nickel post.
Described lysis buffer can be containing 300mmol/L NaCl and pH7.4,100mmol/L of 10mmol/L imidazoles
Tris-HCl buffer.
Described lavation buffer solution can be pH7.4,100mmol/L Tris-HCl buffer containing 300mmol/L NaCl.
In said method, described " culturing engineering bacterium " may also include step (6): under the conditions of 4 DEG C, by described step
Suddenly after the post excessively that (5) obtain, solution carries out desalination of dialysing.
In said method, before carrying out described step (1), also can carry out following steps (A): by described recombinant bacterium
Monoclonal is inoculated in 5mL fluid medium, 35 DEG C～39 DEG C (as 35 DEG C～37 DEG C, 37 DEG C～39 DEG C, 35 DEG C,
37 DEG C or 39 DEG C), 100rpm～140rpm be (such as 100rpm～120rpm, 120rpm～140rpm, 100rpm, 120rpm
Or 140rpm) shaken cultivation 10h～14h (such as 10h～12h, 12h～14h, 10h, 12h or 14h), planted
Sub-liquid；Described seed liquor is inoculated into fluid medium, and inoculum concentration is 1:100 (volume ratio).
Any of the above-described described fluid medium can be the LB fluid medium containing 50 μ g/mL kanamycin.
The protein UGTCs4 utilizing any of the above-described described method to prepare falls within protection scope of the present invention.
The application utilizing the protein UGTCs4 that any of the above-described described method prepares falls within the protection model of the present invention
Enclosing, the application of described protein UGTCs4 is following c1) or c2) or c3) c4) or c5) or c6):
C1) as the application of crocetin glycosyl transferase；
C2) there is the application in the product of crocetin glycosyl transferase function in preparation.
C3) application in catalysis crocetin generation glycosylation；
C4) there is the application in glycosylated product in preparation for being catalyzed crocetin.
C5) application in producing crocin glycosides 5 and/or crocin glycosides 3；
C6) application producing in the product of crocin glycosides 5 and/or crocin glycosides 3 it is used in preparation.
In above-mentioned application, described " producing crocin glycosides 5 and/or crocin glycosides 3 " is using crocetin the end of as
Thing.
It is demonstrated experimentally that utilize the method preparing protein UGTCs4 provided by the present invention can prepare protein UGTCs4,
Protein UGTCs4 has crocetin glycosyl transferase activity, can form crocin by order glycosylation crocetin
Glycosides 5 and crocin glycosides 3.The method of protein UGTCs4 of preparing provided by the present invention is to producing crocin glycosides 5
And/or crocin glycosides 3, preparation have crocetin glycosyl transferase function product or catalysis crocetin occur sugar
Base is respectively provided with significant application value.
Accompanying drawing explanation
Fig. 1 is the pcr amplification product of UGTCs4 gene.
Fig. 2 is the variable concentrations IPTG impact on the expression of restructuring crocetin glycosyl transferase.
Fig. 3 is the impact on the expression of restructuring crocetin glycosyl transferase of the different induction time.
Fig. 4 is the impact on the expression of restructuring crocetin glycosyl transferase of the different cell concentration.
Fig. 5 is the impact on the expression of restructuring crocetin glycosyl transferase of the different inducing temperature.
Fig. 6 is different inducing temperature and the induction time impact on the expression of restructuring crocetin glycosyl transferase.
Fig. 7 is the SDS-PAGE result utilizing ni-sepharose purification crocetin glycosyl transferase solution.
Fig. 8 is the experimental result of 1 in embodiment 2 step 2.
Fig. 9 is the experimental result of 2 in embodiment 2 step 2.
Figure 10 is the experimental result of 3 in embodiment 2 step 2.
Figure 11 is the experimental result of 4 in embodiment 2 step 2.
Figure 12 is the mass spectrum of crocetin.
Figure 13 is the mass spectrum of crocin glycosides 5.
Detailed description of the invention
Below in conjunction with detailed description of the invention, the present invention is further described in detail, the embodiment be given only for
Illustrate the present invention rather than in order to limit the scope of the present invention.
Experimental technique in following embodiment, if no special instructions, is conventional method.
Material used in following embodiment, reagent etc., if no special instructions, the most commercially obtain.
Quantitative test in following example, is respectively provided with three times and repeats experiment, results averaged.
Carrier pET28a (+) it is invitrogen Products；E. coli bl21 (DE3) is sky root biochemistry section
Skill (Beijing) company limited product；Nickel post is U.S.'s GE Products, and catalog number is 04003272-EE.DTT
For Amersco Products, catalog number is 0281.UDPG (UDP-Glucose) is that sigma company produces
Product, catalog number is U4625.Crocetin is Chromedex Products, and catalog number is
ASB00003885-010.C18 reversed-phase column (4.6mm × 250mm) is Japan's GL Sciences Inc product.Efficiently
Chromatograph of liquid and diode array detector are Shimadzu Corporation of Japan product.Ultrasonic Cell Disruptor is Ningbo new sesame biology section
Skill company limited product.
Stigma Croci suspension cell in following embodiment is that Stigma Croci cell suspension is trained with reference to Chen Shuan, Wang Xiaodong etc.
The foundation of the system of supporting and optimization. prepared by the method in biotechnology circular 2010. (07): 157-160..
LB fluid medium: 1g NaCl, 0.5g yeast extract and 1g tryptone are dissolved in 100mL deionized water,
Regulation pH value is 7.0.
Sample-loading buffer is containing 2% (mass volume ratio) SDS, 10% (percent by volume) glycerol and 1% (volume basis
Than) pH6.8,50mM Tris-HCl buffer of beta-mercaptoethanol.
Lysis buffer is pH7.4,100mmol/L Tris-HCl containing 300mmol/L NaCl and 10mmol/L imidazoles
Buffer.
Lavation buffer solution is pH7.4,100mmol/L Tris-HCl buffer containing 300mmol/L NaCl.
By the protein named protein UGTCs4 shown in the sequence 2 of sequence table, its encoding gene such as sequence table
Shown in sequence 1.
Embodiment 1, the preparation of restructuring crocetin glycosyl transferase
One, the structure of recombiant plasmid
1, use Trizol method to extract the total serum IgE of Stigma Croci suspension cell, this total serum IgE reverse transcriptase reverse transcription is gone out
First chain cDNA.
2, synthetic primers F 1:5 '-CCATATG(underscore is restricted to GAGCAGAAAGATGAGAACGGAA-3 '
Restriction endonuclease Nde I recognition sequence) and R1:5 '-CGTCGACTTTACAACAATGATCAATGAACTCCT-3 ' (underscore
For restricted enzyme Sal I recognition sequence).
3, with step 1 obtain cDNA as template, carry out PCR amplification with F1 and R1 of the 2-in-1 one-tenth of step for primer, obtain
The pcr amplification product of about 1388bp.Detect with 1% agarose gel electrophoresis, result see Fig. 1 (M is DNA Marker,
Swimming lane 1 is pcr amplification product, and arrow indication is target PCR amplifications product).
Response procedures: 94 DEG C of 5min；94 DEG C of 30s, 68 DEG C of 30s, 72 DEG C of 1min 30s, totally 35 circulations；72℃
10min。
4, with the pcr amplification product obtained in restricted enzyme Nde I and Sal I double digestion step 3, reclaim about
The fragment of 1386bp.
5, with restricted enzyme Nde I and Sal I double digestion carrier pET28a (+), reclaim the carrier bone of about 5350bp
Frame.
6, the carrier framework that fragment step 4 obtained obtains with step 5 is connected, and obtains recombiant plasmid pET28a-
UGTCs4。
According to sequencing result, recombiant plasmid pET28a-UGTCs4 is carried out structure and is described as follows: by carrier pET28a (+)
Nde I recognition sequence and Sal I recognition sequence between DNA small fragment to replace with nucleotide sequence be sequence 1 in sequence table
Shown DNA molecular.In recombiant plasmid pET28a-UGTCs4, the DNA molecular shown in sequence 1 of sequence table and carrier bone
The coded sequence of the His-tag label (being made up of 6 histidine residues) on frame merges, shown in the sequence 3 of formation sequence table
Fusion gene, the protein UGTCs4 with His-tag label shown in the sequence 4 of expressed sequence table, have
The protein UGTCs4 of His-tag label is restructuring crocetin glycosyl transferase.The egg shown in sequence 4 of sequence table
The expection molecular weight of white matter is 54.8KD.
Two, the groping of optimum condition of the expression of restructuring crocetin glycosyl transferase
1, the variable concentrations IPTG impact on the expression of restructuring crocetin glycosyl transferase
1. recombiant plasmid pET28a-UGTCs4 step one built imports escherichia coli Rosetta (DE3), obtains
Recombinant bacterium, by named for this recombinant bacterium Rosetta (DE3)-pET28a-UGTCs4.
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in 5mL containing 50 μ g/mL kanamycin
LB fluid medium, 37 DEG C, 120rpm shaken cultivation 12h, obtain cultivate bacterium solution 1.Take cultivation bacterium solution 1 0.3mL,
It is inoculated in the 30mL LB fluid medium containing 50 μ g/mL kanamycin, 37 DEG C, 180rpm with 1:100 (volume ratio)
Shaken cultivation is to OD600Value 0.3～0.4, obtains cultivating bacterium solution 2.
3. complete step 2. after, take cultivation bacterium solution 2, centrifugal, it is thus achieved that the thalline before IPTG induction.
4. complete step 2. after, bacterium solution 2 add IPTG obtain induction system to cultivating, IPTG is in induction system
Concentration be 50 μMs, 100 μMs, 200 μMs, 300 μMs, 400 μMs, 500 μMs, 600 μMs or 700 μMs, so
Latter 37 DEG C, 120rpm shaken cultivation 2.5h, centrifugal collection thalline, it is thus achieved that the thalline after IPTG induction.
Take the thalline before IPTG induction and the thalline after IPTG induction, addition LB fluid medium to OD the most respectively600
Value adjusts to unanimously, 25 DEG C, 9000rpm be centrifuged 1min, abandon supernatant, collect thalline.
6. taking the thalline that 5. step obtains, add sample-loading buffer, boiling water bath boils 13min；Then 25 DEG C, 9000rpm
Centrifugal 10min, collects supernatant and carries out SDS-PAGE.
Experimental result is shown in that (M is albumen marker to Fig. 2, and 1 is the thalline before IPTG induction, after 2 are 50 μMs of IPTG inductions
Thalline, 3 is the thalline after 100 μMs of IPTG induction, and 4 is the thalline after 200 μMs of IPTG inductions, and 5 is 300 μMs of IPTG
Thalline after induction, 6 is the thalline after 400 μMs of IPTG inductions, and 7 is the thalline after 500 μMs of IPTG inductions, and 8 is 600
Thalline after μM IPTG induction, 9 is the thalline after 700 μMs of IPTG inductions, and arrow indication is target protein).Result
Showing, the optimal IPTG induced concentration of the expression of restructuring crocetin glycosyl transferase is 50 μMs～600 μMs；Work as IPTG
When induced concentration is 700 μMs, the expression of restructuring crocetin glycosyl transferase has a certain degree of suppression.
2, the different IPTG induction times impact on the expression of restructuring crocetin glycosyl transferase
1. with in step 1 1..
2. with in step 1 2..
3. with in step 1 3..
4. complete step 2. after, complete step 2. after, bacterium solution 2 add IPTG obtain induction system, IPTG to cultivating
Concentration in induction system is 200 μMs, then 37 DEG C, 120rpm shaken cultivation, incubation time is 30min, 1h,
1.5h, 2h, 2.5h, 3h or 4h, centrifugal collection thalline, it is thus achieved that the thalline after IPTG induction.
5. with in step 1 5..
6. with in step 1 6..
Experimental result is shown in that (M is albumen marker to Fig. 3, and 1 is the thalline before IPTG induction, after 2 for IPTG induction 30min
Thalline, 3 is the thalline after IPTG induction 1h, and 4 is the thalline after IPTG induction 1.5h, and 5 is the thalline after IPTG induction 2h,
6 is the thalline after IPTG induction 2.5h, and 7 is the thalline after IPTG induction 3h, and 8 is the thalline after IPTG induction 4h, arrow
Indication is target protein).Result shows, the optimal IPTG induction time of the expression of restructuring crocetin glycosyl transferase is
30min～4h.
3, the different cell concentrations impact on the expression of restructuring crocetin glycosyl transferase
1. with in step 1 1..
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in 5mL containing 50 μ g/mL kanamycin
LB fluid medium, 37 DEG C, 120rpm shaken cultivation 12h, obtain cultivate bacterium solution 1.Take cultivation bacterium solution 1 0.3mL,
It is inoculated in the 30mL LB fluid medium containing 50 μ g/mL kanamycin, 37 DEG C, 180rpm with 1:100 (volume ratio)
Shaken cultivation, obtains cultivating bacterium solution 2, cultivates the OD of bacterium solution 2600Value is 0.32,0.41,0.54,0.63 or 0.76.
3. with in step 1 3..
4. complete step 2. after, bacterium solution 2 add IPTG obtain induction system to cultivating, IPTG is in induction system
Concentration be 200 μMs, then 37 DEG C, 120rpm shaken cultivation 2.5h, centrifugal collect thalline, it is thus achieved that IPTG induces
After thalline.
5. with in step 1 5..
6. with in step 1 6..
Experimental result is shown in that (M is albumen marker to Fig. 4, and 1 is the thalline before IPTG induction, and 2 is OD600Value is the training of 0.32
Thalline after bacteria liquid 2IPTG induction, 3 is OD600Value is the thalline after the cultivation bacterium solution 2IPTG induction of 0.41, and 4 is OD600
Value is the thalline after the cultivation bacterium solution 2IPTG induction of 0.54, and 5 is OD600After value is the cultivation bacterium solution 2IPTG induction of 0.63
Thalline, 6 is OD600Value is the thalline after the cultivation bacterium solution 2IPTG induction of 0.76, and arrow indication is target protein).Result
Show, the OD of the optimal cell concentration of the expression of restructuring crocetin glycosyl transferase600Value is 0.3～0.8.
4, the different inducing temperatures impact on the expression of restructuring crocetin glycosyl transferase
(1) the inducing temperature 28 DEG C impact on the expression of restructuring crocetin glycosyl transferase
1. with in step 1 1..
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in 5mL containing 50 μ g/mL kanamycin
LB fluid medium, 37 DEG C, 120rpm shaken cultivation 12h, obtain cultivate bacterium solution 1.Take cultivation bacterium solution 1 0.3mL,
It is inoculated in the 30mL LB fluid medium containing 50 μ g/mL kanamycin, 28 DEG C, 120rpm with 1:100 (volume ratio)
Shaken cultivation is to OD600Value 0.3～0.4, obtains cultivating bacterium solution 2.
3. with in step 1 3..
4. complete step 2. after, bacterium solution 2 add IPTG obtain induction system to cultivating, IPTG is in induction system
Concentration be 200 μMs, then 28 DEG C, 120rpm shaken cultivation, incubation time is 3h, 4h, 5h, 6h, 7h,
8h or 9h, centrifugal collection thalline, it is thus achieved that the thalline after IPTG induction.
5. with in step 1 5..
6. with in step 1 6..
Experimental result is shown in that (M is protein low-molecular-weight marker to Fig. 5, and 1 is the thalline before IPTG induction, and 2 is IPTG
Thalline after induction 3h, 3 is the thalline after IPTG induction 4h, and 4 is the thalline after IPTG induction 5h, and 5 is IPTG
Thalline after induction 6h, 6 is the thalline after IPTG induction 7h, and 7 is the thalline after IPTG induction 8h, and 8 is IPTG
Thalline after induction 9h, arrow indication is target protein).Result shows, restructuring crocetin glycosyl transferase is main
It is present in inclusion body, it is seen that the temperature of the expression of restructuring crocetin glycosyl transferase can not be 28 DEG C.
(2) the inducing temperature 37 DEG C impact on the expression of restructuring crocetin glycosyl transferase
According to the method for step (1), replacing with 37 DEG C by 28 DEG C, other step is the most constant, result restructuring Stigma Croci
Acid glycosyl transferase is also primarily present in inclusion body, it is seen that the temperature of the expression of restructuring crocetin glycosyl transferase can not
It it is 37 DEG C.
(3) the inducing temperature 18 DEG C impact on the expression of restructuring crocetin glycosyl transferase
1. with in step 1 1..
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in 5mL containing 50 μ g/mL kanamycin
LB fluid medium, 37 DEG C, 120rpm shaken cultivation 12h, obtain cultivate bacterium solution 1.Take cultivation bacterium solution 1 0.3mL,
It is inoculated in 30mL containing 50 μ g/mL kanamycin LB fluid mediums, 18 DEG C, 120rpm with 1:100 (volume ratio)
Shaken cultivation is to OD600Value 0.3～0.4, obtains cultivating bacterium solution 2.
3. with in step 1 3..
4. complete step 2. after, bacterium solution 2 add IPTG obtain induction system to cultivating, IPTG is in induction system
Concentration be 200 μMs, then 18 DEG C, 120rpm shaken cultivation, incubation time is 6h, 8h, 10h, 12h, 14h,
16h or 18h, centrifugal collection thalline, it is thus achieved that the thalline after IPTG induction.
5. with in step 1 5..
6. with in step 1 6..
Experimental result is shown in that (M is albumen marker to Fig. 6, and 1 is the thalline before IPTG induction, and 2 is the bacterium after IPTG induction 6h
Body, 3 is the thalline after IPTG induction 8h, and 4 is the thalline after IPTG induction 10h, and 5 is the thalline after IPTG induction 12h,
6 is the thalline after IPTG induction 14h, and 7 is the thalline after IPTG induction 16h, and 8 is the thalline after IPTG induction 18h, arrow
Head indication is target protein).Result shows, 18 DEG C, IPTG induction 6h～18h can induce restructuring crocetin glycosyl turn
Move the expression of enzyme.
Three, the expression and purification of restructuring crocetin glycosyl transferase
1. recombiant plasmid pET28a-UGTCs4 is imported escherichia coli Rosetta (DE3), obtain recombinant bacterium, should
Recombinant bacterium named Rosetta (DE3)-pET28a-UGTCs4.
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in 5mL containing 50 μ g/mL kanamycin
LB fluid medium, 37 DEG C, 120rpm shaken cultivation 12h, obtain cultivate bacterium solution 1.Take cultivation bacterium solution 1 0.3mL,
It is inoculated in 30mL containing 50 μ g/mL kanamycin LB fluid mediums, 18 DEG C, 120rpm with 1:100 (volume ratio)
Shaken cultivation is to OD600Value 0.3～0.4, obtains cultivating bacterium solution 2.
3. complete step 2. after, take cultivation bacterium solution 2,5000rpm is centrifuged 10min, collects thalline, it is thus achieved that IPTG lures
The thalline of leading.
4. complete step 2. after, bacterium solution 2 add IPTG obtain induction system to cultivating, IPTG is in induction system
Concentration be 200 μMs, then 18 DEG C, 120rpm shaken cultivation 16h, 5000rpm is centrifuged 10min, collects thalline,
Obtain the thalline after IPTG induction.
5. take the thalline after IPTG induction, washed once with lysis buffer.
6. take into step thalline 5., with 1:10 (volume ratio) add lysis buffer, put into-20 DEG C frozen,
Obtain frozen bacterium solution.
7. take into step frozen bacterium solution 6., after thawing rapidly, put ultrasonication on Ultrasonic Cell Disruptor on ice (super
Acoustic power 100W, cyclic program is: broken 6s, stops 4s, totally 120 circulations), then 4 DEG C, 13000rpm
Centrifugal 30min, collects supernatant, the bacterial cell disruption supernatant after named IPTG induction.
According to 5. to step 7., the thalline after being induced by IPTG replaces with the thalline before IPTG induction, obtains IPTG
Bacterial cell disruption supernatant before induction.
The most first with the lysis buffer balance nickel post (flow velocity is 1mL/min) of 5～10 column volumes, then loading step
7. the bacterial cell disruption supernatant (flow velocity is 1mL/min) after the IPTG induction obtained, more successively with 5 column volumes
Lavation buffer solution, the lavation buffer solution containing 80mmol/L imidazoles of 5 column volumes and 5 containing 20mmol/L imidazoles
The lavation buffer solution containing 100mmol/L imidazoles of column volume rinses nickel post, to remove most foreign protein, finally uses
The lavation buffer solution containing 250mmol/L imidazoles of 5 column volumes rinse nickel post and collected post after solution, be restructuring
Crocetin glycosyl transferase solution.
Bacterial cell disruption supernatant after IPTG is induced, the bacterial cell disruption supernatant before IPTG induction and step 8. in each
The eluting of elution step is collected liquid and is carried out SDS-PAGE.
Experimental result see Fig. 7 (M is protein molecular weight Marker, 1 be IPTG induction before bacterial cell disruption supernatant,
2 is the bacterial cell disruption supernatant after IPTG induction, and 3 is that the lavation buffer solution containing 20mmol/L imidazoles carries out washing of eluting
De-liquid of collecting, 4 is the eluting collection liquid that the lavation buffer solution containing 80mmol/L imidazoles carries out eluting, and 5 for containing
The lavation buffer solution of 100mmol/L imidazoles carries out the eluting of eluting and collects liquid, and 6 is that the washing containing 250mmol/L imidazoles is delayed
Rush liquid and carry out the eluting collection liquid of eluting).Result shows, restructuring crocetin glycosyl transferase solution shows single molecule
Amount band, corresponding molecular weight is 54.8KDa, consistent with expection molecular weight.
The content of restructuring crocetin glycosyl transferase in bacterial cell disruption supernatant after the IPTG induction that 7. step obtains
Being 0.03 μ g/ μ L, crocetin glycosyl of recombinating in the restructuring crocetin glycosyl transferase solution that 8. step obtains turns
The content moving enzyme is 2.13 μ g/ μ L.Restructuring safranine in bacterial cell disruption supernatant after the IPTG induction that 7. step obtains
The computational methods of the content of flower acid glycosyl transferase are: the total protein content of detection liquid-phase system, then by albumen
Each band in electrophoresis carries out gray scale scanning and calculates the ratio that restructuring crocetin glycosyl transferase is shared in total protein
Example, is calculated the content of restructuring crocetin glycosyl transferase.The restructuring crocetin glycosyl transfer that 8. step obtains
In enzymatic solution, the content of restructuring crocetin glycosyl transferase is measured according to Bradford method.
Embodiment 2, utilize HPLC detection differential responses under the conditions of recombinate crocetin glycosyl transferase glycosyl transferase
Activity
One, dialysis
Under the conditions of 4 DEG C, restructuring crocetin glycosyl transferase solution embodiment 1 obtained carries out desalination of dialysing,
Replace again in pH7.5,5mM Tris-HCl buffer, finally utilize the super filter tube that molecular weight is 30MW to carry out ultrafiltration
Protein concentrate, it is thus achieved that purity is the protein sample of the restructuring crocetin glycosyl transferase of 98%.This protein sample is entered
The order-checking of row 25 amino acid residues of N end, result shows, 25 amino acid whose sequence such as sequence tables of this albumen n end
Middle sequence 4 is from N end shown in the 1st to 25.
Two, utilize HPLC detection differential responses under the conditions of recombinate crocetin glycosyl transferase glycosyl transferase activity
1, the differential responses time impact on the glycosyl transferase activity of restructuring crocetin glycosyl transferase
(1) preparing the 200 μ L restructuring external enzymatic reaction systems of crocetin glycosyl transferase, this system solvent is
PH7.5,50mM Tris-HCl buffer, solute be 1mM DTT, 5mM UDPG (UDP-Glucose), 50
μM MgCl2, the restructuring crocetin glycosyl transferase that obtains of 10 μMs of crocetins and 16 μ g steps one.
(2) under the conditions of system prepared by step (1) being placed in 37 DEG C, react the most respectively 0min, 30min, 1h,
1.5h, 2h, 2.5h, 3h or 4h.
(3) after completing step (2), adding 400 μ L hplc grade methanols and terminate reaction, then 13000rpm is centrifuged
10min, collects supernatant the organic membrane filtration through 0.45 μm, obtains crocetin glycosyl transferase of recombinating
The testing sample of external enzymatic reaction.
(4) take the external enzymatic reaction of the restructuring crocetin glycosyl transferase that step (3) obtains testing sample and
Crocetin standard solution (dissolving crocetin to its concentration with methanol is 10 μMs), carries out the inspection of HPLC-MS
Survey.Use is furnished with the high performance liquid chromatograph of C18 reversed-phase column (4.6mm × 150mm) and UV-vis detector is carried out
HPLC detects.Flowing is made up of methanol (A) and water (B), and flow velocity is 1mL/min, uses following gradient elution
Condition: in 0～20min, in flowing mutually, the volumn concentration of methanol is at the uniform velocity increased to 50% by 20%, the volume of water
Percentage composition is at the uniform velocity down to 50% by 80%, carries out linear gradient elution；In 20～50min, methanol in flowing mutually
Volumn concentration is at the uniform velocity increased to 70% by 50%, and the volumn concentration of water is at the uniform velocity down to 30% by 50%, carries out linear
Gradient elution；In 50～55min, enter by the flowing phase 1 being mixed to get according to the volume ratio of 7:3 by first alcohol and water
Row eluting；In 55～60min, carry out eluting with methanol；In 60～65min, the volume of methanol in flowing mutually
Percentage composition is at the uniform velocity down to 20% by 100%, and the volumn concentration of water is at the uniform velocity risen to 80% by 0%, carries out linear gradient
Eluting；In 65～70min, 2 wash mutually with the flowing being mixed to get according to the volume ratio of 2:8 by first alcohol and water
De-.Detection wavelength is 440nm.(methanol dissolves crocetin to it to Simultaneous Quantitative Analysis crocetin standard solution
Concentration is 10 μMs).Mass Spectrometer Method condition is: scanning of the mass spectrum scope 100-2000 (m/z)；Carrier gas: High Purity Nitrogen
Gas, flow rate of carrier gas: 40arb, secondary air speed: 2arb, blowback air flow velocity: 1arb, capillary temperature:
275 DEG C, capillary voltage: 100V, spray voltage: 3.2V.Experiment in triplicate, repeats to set 10 every time
Triangular flask.
The experimental result of the testing sample HPLC detection of the external enzymatic reaction of restructuring crocetin glycosyl transferase is shown in figure
8 (CK is crocetin standard substance, and abscissa is retention time), under this chromatographic condition, the reservation of crocetin
Time is 35min, and the retention time of crocin glycosides 5 is 25min.The high resolution mass spectrum experimental result of crocetin
Seeing Figure 12, the high resolution mass spectrum experimental result of crocin glycosides 5 is shown in Figure 13.Result shows, the response time is to restructuring
The glycosyl transferase activity impact of crocetin glycosyl transferase is more notable, along with the prolongation in response time, safranine
Flower acid concentration and crocin glycosides 3 gradually decrease, and crocin glycosides 5 is gradually increased.Therefore, restructuring crocetin
Glycosyl transferase has order glycosylation crocetin and forms crocin glycosides 3 and the function of crocin glycosides 5, implements
The restructuring crocetin glycosyl transferase of example 1 preparation has the glycosyl transferase activity with crocetin as substrate.
2, the different pH value impact on the glycosyl transferase activity of restructuring crocetin glycosyl transferase
(1) preparing the 200 μ L restructuring external enzymatic reaction systems of crocetin glycosyl transferase, this system solvent is
PH6.8,50mM Tris-HCl buffer, pH7.5,50mM Tris-HCl buffer, pH8.0,50mM Tris-HCl
Buffer or pH8.8,50mM Tris-HCl buffer, solute be 1mM DTT, 5mM UDPG (UDP-Glucose),
50μM MgCl2, the restructuring crocetin glycosyl transferase that obtains of 10 μMs of crocetins and 16 μ g steps one.
(2), under the conditions of system prepared by step (1) being placed in 37 DEG C, 30min is reacted.
(3) with (3) in step 1.
(4) with (4) in step 1.
The experimental result of the testing sample HPLC detection of the external enzymatic reaction of restructuring crocetin glycosyl transferase is shown in figure
9 (CK is crocetin standard substance, and abscissa is retention time), under this chromatographic condition, the reservation of crocetin
Time is 35min.Result shows, the different pH value glycosyl transferase activity to restructuring crocetin glycosyl transferase
Impact is more notable, and when pH value is 7.5, the glycosyl transferase activity of restructuring crocetin glycosyl transferase is the highest.
3, the impact of the glycosyl transferase activity of different metal ion pair restructuring crocetin glycosyl transferase
(1) preparing the 200 μ L restructuring external enzymatic reaction systems of crocetin glycosyl transferase, this system solvent is
PH7.5,50mM Tris-HCl buffer, solute be 1mM DTT, 5mM UDPG (UDP-Glucose), 50
The restructuring crocetin glycosyl transferase that μM chlorate, 10 μMs of crocetins and 16 μ g steps one obtain.Chlorine
Change salt is MgCl2、CaCl2、MnCl2、CuCl2, NaCl or ZnCl2。
(2), under the conditions of system prepared by step (1) being placed in 37 DEG C, 30min is reacted.
(3) with (3) in step 1.
(4) with (4) in step 1.
The experimental result of the testing sample HPLC detection of the external enzymatic reaction of restructuring crocetin glycosyl transferase is shown in figure
10 (CK is crocetin standard substance, and abscissa is retention time) under this chromatographic condition, the reservation of crocetin
Time is 35min.Result shows, the glycosyl transferase of different metal ion pair restructuring crocetin glycosyl transferase is lived
Property impact the most notable: Cu2+(with CuCl2Form add), Zn2+(with ZnCl2Form adds) or Mn2+(with
MnCl2Form add) to restructuring crocetin glycosyl transferase glycosyl transferase activity have obvious inhibitory action；
Mg2+(with MgCl2Form add), Ca2+(with Ca Cl2Form adds) or Na+(adding with NaCl form) is right
The glycosyl transferase activity of restructuring crocetin glycosyl transferase has obvious facilitation, can promote crocetin of recombinating
Glycosyl transferase order glycosylation crocetin forms crocin glycosides 3 and crocin glycosides 5.
4, the different concentration of substrate impact on the glycosyl transferase activity of restructuring crocetin glycosyl transferase
(1) preparing the 200 μ L restructuring external enzymatic reaction systems of crocetin glycosyl transferase, this system solvent is
PH7.5,50mM Tris-HCl buffer, solute be 1mM DTT, 5mM UDPG (UDP-Glucose), 50
μM MgCl2, the restructuring crocetin glycosyl transferase that obtains of crocetin and 16 μ g steps one.Crocetin exists
Concentration in this system is 10 μMs, 20 μMs, 30 μMs, 40 μMs, 50 μMs or 60 μMs.
(2), under the conditions of system prepared by step (1) being placed in 37 DEG C, 30min is reacted.
(3) with (3) in step 1.
(4) with (4) in step 1.
The experimental result of the testing sample HPLC detection of the external enzymatic reaction of restructuring crocetin glycosyl transferase is shown in figure
11 (CK is crocetin standard substance, and abscissa is retention time) under this chromatographic condition, the reservation of crocetin
Time is 35min.Result shows, the glycosyl transferase of restructuring crocetin glycosyl transferase is lived by different concentration of substrate
Property impact the most notable: when concentration of substrate is 10 μMs, restructuring crocetin glycosyl transferase glycosyl transferase live
Property is the highest；Along with the increase of concentration of substrate, crocin glycosides 3 constantly accumulates, and crocin glycosides 5 constantly reduces.
Claims (10)
1. the method preparing protein UGTCs4, comprises the steps: culturing engineering bacterium, obtains described albumen
Matter UGTCs4；Described engineering bacteria is that recombinant expression carrier imports the recombinant bacterium that Host Strains obtains；Described recombinant expressed
Carrier is that the encoding gene of described protein UGTCs4 is inserted the recombiant plasmid that expression vector obtains；
Described protein UGTCs4 is following a1) or a2) or a3) or a4):
A1) protein shown in sequence 2 during aminoacid sequence is sequence table；
A2) in sequence table the N end of the protein shown in sequence 2 or/and C end connects the fused protein that obtains of label；
A3) protein shown in sequence 4 during aminoacid sequence is sequence table；
A4) by a1) or a2) or a3) shown in protein through one or several amino acid residue replacement and/or
The protein with crocetin glycosyl transferase activity that disappearance and/or interpolation obtain.
2. the method for claim 1, it is characterised in that: the encoding gene of described protein UGTCs4 is such as
Lower b1) or b2) or b3) or b4) shown in DNA molecular:
B1) DNA molecular shown in sequence 1 during nucleotide sequence is sequence table；
B2) DNA molecular shown in sequence 3 during nucleotide sequence is sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or more than 75% homogeneity, and coding right is wanted
Seek the DNA molecular of protein UGTCs4 described in 1；
B4) under strict conditions with b1) or b2) institute in the nucleotide sequence hybridization that limits, and coding claim 1
State the DNA molecular of protein UGTCs4.
3. method as claimed in claim 1 or 2, it is characterised in that: described recombinant expression carrier is to express load
The recombiant plasmid that the DNA molecular shown in sequence 1 of the multiple clone site insertion sequence table of body obtains.
4. the method for claim 1, it is characterised in that: described Host Strains is escherichia coli.
5. the method as described in Claims 1-4 is arbitrary, it is characterised in that: the process of described " culturing engineering bacterium "
In, carry out IPTG induction.
6. method as claimed in claim 5, it is characterised in that: the method for described IPTG induction is as follows: work as cultivation
The OD of system600nmWhen value is 0.3～0.4, adds IPTG and to make its concentration in cultivating system be 50 μMs～600
μM。
7. method as claimed in claim 5, it is characterised in that: described " culturing engineering bacterium " comprises the steps:
(1) cultivate under the conditions of 18 DEG C～20 DEG C, until the OD of cultivating system600nmValue is 0.3～0.4；
(2), after completing step (1), add IPTG and to make its concentration in cultivating system be 50 μMs～600 μMs,
Cultivate 6h～18h for 18 DEG C～20 DEG C.
8. the protein UGTCs4 that the arbitrary described method of claim 1 to 7 prepares.
9. the application of the protein UGTCs4 described in claim 8, for following c1) or c2) or c3) or c4)
Or c5) or c6):
C1) as the application of crocetin glycosyl transferase；
C2) there is the application in the product of crocetin glycosyl transferase function in preparation.
C3) application in catalysis crocetin generation glycosylation；
C4) there is the application in glycosylated product in preparation for being catalyzed crocetin.
C5) application in producing crocin glycosides 5 and/or crocin glycosides 3；
C6) application producing in the product of crocin glycosides 5 and/or crocin glycosides 3 it is used in preparation.
Apply the most as claimed in claim 9, it is characterised in that: described production crocin glycosides 5 and/or Stigma Croci
Element glycosides 3 is using crocetin as substrate.
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| CN106906192A (en) * | 2017-04-17 | 2017-06-30 | 南京工业大学 | Glucosyltransferase and application thereof in synthesis of crocetin glucose ester |
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| CN110004123A (en) * | 2019-04-18 | 2019-07-12 | 安徽农业大学 | A kind of tea tree sucrose synthase CsSUS2, preparation method and application |
| CN110791512A (en) * | 2018-08-02 | 2020-02-14 | 中国医学科学院药用植物研究所 | Screening identification and application of glycosyltransferases GjUGT94E13 and GjUGT74F8 involved in crocin synthesis |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106906192A (en) * | 2017-04-17 | 2017-06-30 | 南京工业大学 | Glucosyltransferase and application thereof in synthesis of crocetin glucose ester |
| CN106906192B (en) * | 2017-04-17 | 2020-10-30 | 南京工业大学 | Glucosyltransferase and application thereof in synthesis of crocetin glucose ester |
| IT201700089843A1 (en) * | 2017-08-03 | 2019-02-03 | Enea Agenzia Naz Per Le Nuove Tecnologie Lenergia E Lo Sviluppo Economico Sostenibile | Genes and methods for the production and biotechnological compartmentation of high added value apocarotenoids |
| CN110791512A (en) * | 2018-08-02 | 2020-02-14 | 中国医学科学院药用植物研究所 | Screening identification and application of glycosyltransferases GjUGT94E13 and GjUGT74F8 involved in crocin synthesis |
| CN110791512B (en) * | 2018-08-02 | 2022-05-17 | 中国医学科学院药用植物研究所 | Screening identification and application of glycosyltransferases GjUGT94E13 and GjUGT74F8 involved in crocin synthesis |
| CN109943547A (en) * | 2019-04-18 | 2019-06-28 | 安徽农业大学 | A kind of tea tree sucrose synthase CsSUS587, preparation method and application |
| CN110004123A (en) * | 2019-04-18 | 2019-07-12 | 安徽农业大学 | A kind of tea tree sucrose synthase CsSUS2, preparation method and application |
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