CN105950580B - A method of preparing protein UGTCs4 - Google Patents
A method of preparing protein UGTCs4 Download PDFInfo
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- CN105950580B CN105950580B CN201610355947.3A CN201610355947A CN105950580B CN 105950580 B CN105950580 B CN 105950580B CN 201610355947 A CN201610355947 A CN 201610355947A CN 105950580 B CN105950580 B CN 105950580B
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Abstract
The invention discloses a kind of method for preparing protein UGTCs4, protein UGTCs4 has crocetin glycosyl transferase activity.Method provided by the present invention includes the following steps: culturing engineering bacterium, obtains the protein UGTCs4；The engineering bacteria is the recombinant bacterium for obtaining recombinant expression carrier importing host strain；The recombinant expression carrier is that the encoding gene of the protein UGTCs4 is inserted into the recombinant plasmid that expression vector obtains.Experiments have shown that, crocetin can be sequentially glycosylated using protein UGTCs4 prepared by method provided by the present invention and forms crocin glycosides 5 and crocin glycosides 3, be there is the product of crocetin glycosyl transferase function or catalysis crocetin glycosylation occurs production crocin glycosides 5 and/or crocin glycosides 3, preparation and all had significant application value.
Description
Technical field
The invention belongs to field of biotechnology, and in particular to a method of prepare protein UGTCs4.
Background technique
Safflower is called safron, west safflower, is perennial irides safron (Crocus sativus L.)
Drying column cap, be a kind of rare Chinese medicine in China.The total glycosides of safflower is the pigment compound extracted from safflower, master
Wanting ingredient is crocin (crocus), crocetin (crocetin) and its derivative, and wherein crocin is crocetin
A series of ester glycosides being combined into different sugar, are one of main actives of safflower, have platelet aggregation-against, anti-blood
The multiple pharmacological effects such as bolt forms, resists myocardial ischemia, anticancer, and almost non-toxic side effect.Existing research shows that crocetin
Glycosyl transferase has the function of that glycosylating crocetin forms crocin, such as Morgan in 2004 is from safflower style
There is glycosylation crocetin to form hiding for separation discovery UGTCs2 gene (GeneID:AY36026), the crude protein of prokaryotic expression
The function of carthamin, therefore be the encoding gene of crocetin glycosyl transferase by UGTCs2 gene annotation；For another example Mai in 2012
Nagatoshi separates discovery UGT75L6 gene (GeneID:AB555731) and UGT94E5 gene from cape jasmine cell
(GeneID:555739), there is the protein crude extract administration of UGT75L6 gene prokaryotic sequence glycosylation crocetin to form safranine
The protein crude extract administration of the function of florigen glycosides 5, crocin glycosides 3 and crocin glycosides 4, UGT94E5 gene prokaryotic has sugar
Base crocin glycosides 5 forms the function of crocin glycosides 2 and crocin glycosides 1, therefore, by UGT75L6 gene and UGT94E5
The encoding gene that gene also annotates as crocetin glycosyl transferase.But it so far, can not still obtain the safranine of high-purity
Spend sour glycosyl transferase.
Summary of the invention
The technical problem to be solved by the present invention is to how prepare the protein with crocetin glycosyl transferase activity.
In order to solve the above technical problems, present invention firstly provides a kind of method for preparing protein UGTCs4, the egg
White matter UGTCs4 has crocetin glycosyl transferase activity.
The method provided by the present invention for preparing protein UGTCs4, it may include following steps: culturing engineering bacterium obtains institute
State protein UGTCs4；The engineering bacteria is the recombinant bacterium for obtaining recombinant expression carrier importing host strain；The recombinant expression
Carrier is that the encoding gene of the protein UGTCs4 is inserted into the recombinant plasmid that expression vector obtains；
The protein UGTCs4 can be following a1) or a2) or a3) or a4):
A1) amino acid sequence is protein shown in sequence 2 in sequence table；
A2) the fused protein that the N-terminal of protein shown in sequence 2 or/and C-terminal connection label obtain in sequence table；
A3) amino acid sequence is protein shown in sequence 4 in sequence table；
A4) by a1) or a2) or a3) shown in protein pass through one or several amino acid residues substitution and/or missing
And/or the protein with crocetin glycosyl transferase activity that addition obtains.
Wherein, sequence 2 can be made of 459 amino acid residues in sequence table；Sequence 4 can be by 492 amino in sequence table
Sour residue composition.
In order to make a1) in protein convenient for purifying, can in sequence table the amino terminal of protein shown in sequence 2 or
Carboxyl terminal connects upper label as shown in Table 1.
The sequence of table 1, 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, the substitution and/or deletion and/or addition of one or several amino acid residues be
No more than the substitution and/or deletion and/or addition of 10 amino acid residues.
Above-mentioned a4) in protein can be artificial synthesized, can also first synthesize its encoding gene, then carry out biological expression and obtain.
Above-mentioned a4) in the encoding gene of protein can be by will be shown in sequence 3 in sequence 1 in sequence table or sequence table
The codon of one or several amino acid residues is lacked in DNA sequence dna, and/or the missense of one or several base-pairs of progress is dashed forward
Become, and/or is obtained in the coded sequence that its 5 ' end and/or 3 ' ends connect label shown in table 1.
In the above method, the encoding gene of the protein UGTCs4 concretely following b1) or b2) or b3) or b4) institute
The DNA molecular shown:
B1) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table；
B2) nucleotide sequence is DNA molecular shown in sequence 3 in sequence table；
B3) and b1) or b2) nucleotide sequence that limits has 75% or 75% or more identity, and encodes claim 1
Described in protein UGTCs4 DNA molecular；
B4) the nucleotide sequence hybridization limited under strict conditions with b1) or b2), and encode egg described in claim 1
The DNA molecular of white matter UGTCs4.
Wherein, the nucleic acid molecules can be DNA, such as cDNA, genomic DNA or recombinant DNA；The nucleic acid molecules can also
To be RNA, such as mRNA or hnRNA.
Wherein, sequence 1 is made of 1377 nucleotide in sequence table, the nucleotide coding sequence table of sequence 1 in sequence table
Amino acid sequence shown in middle sequence 2；Sequence 3 is made of 1476 nucleotide in sequence table, the nucleotide of sequence 3 in sequence table
Amino acid sequence shown in sequence 4 in polynucleotide.
Those of ordinary skill in the art can easily adopt by known method, such as the side of directed evolution and point mutation
Method is mutated the nucleotide sequence of code for said proteins UGTCs4 of the invention.Those have by manually modified
The nucleotide sequence 75% of the isolated protein UGTCs4 or the nucleotide of higher identity with the present invention, as long as
Code for said proteins UGTCs4 is derived from nucleotide sequence of the invention and to be equal to sequence of the invention.
Term " identity " used herein refers to the sequence similarity with native sequence nucleic acid." identity " includes and this hair
The nucleotide sequence of the protein of the composition of amino acid sequence shown in the sequence 2 of bright polynucleotide or the sequence 4 of sequence table
Core with 75% or higher or 80% or higher or 85% or higher or 90% or higher or 95% or higher identity
Nucleotide sequence.Identity can with the naked eye or computer software is evaluated.Using computer software, two or more sequences it
Between identity can be indicated with percentage (%), can be used to evaluate identity between correlated series.
In the above method, the recombinant expression carrier can be in the sequence of the multiple cloning sites insetion sequence table of expression vector
The recombinant plasmid that DNA molecular shown in 1 obtains.
The expression vector can be carrier pET-28a (+).
In the above method, the recombinant expression carrier concretely in I recognition site of Nde of carrier pET-28a (+) and
The recombinant plasmid pET28a-UGTCs4 that DNA molecular shown in the sequence 1 of insetion sequence table obtains between I recognition site of Sal.Weight
Protein shown in the sequence 4 of group plasmid pET28a-UGTCs4 expressed sequence table.
In the above method, the host strain can be Escherichia coli.
The Escherichia coli can be E. coli BL21 (DE3).
In the above method, during " the culturing engineering bacterium ", IPTG induction is carried out.
In the above method, the method for " the IPTG induction " is as follows: as the OD of cultivating system600nmWhen value is 0.3~0.4,
Be added IPTG and make its in cultivating system 50 μM~600 μM of concentration (such as 50 μM, 100 μM, 200 μM, 300 μM, 400 μM,
500 μM, 600 μM, 50 μM~100 μM, 50 μM~200 μM, 50 μM~300 μM, 50 μM~400 μM, 50 μM~500 μM, 50 μM
~600 μM, 100 μM~200 μM, 100 μM~300 μM, 100 μM~400 μM, 100 μM~500 μM, 100 μM~600 μM, 200 μ
M~300 μM, 200 μM~400 μM, 200 μM~500 μM, 200 μM~600 μM, 300 μM~400 μM, 300 μM~500 μM, 300
μM~600 μM, 400 μM~500 μM, 400 μM~600 μM or 500 μM~600 μM).
" IPTG is added and makes its 50 μM~600 μM of concentration in cultivating system " refers to that the IPTG is being trained
Initial concentration in the system of supporting.
In the above method, " the culturing engineering bacterium " may include following steps:
(1) it is cultivated under the conditions of 18 DEG C~20 DEG C (such as 18 DEG C or 20 DEG C), until the OD of cultivating system600nmValue is
0.3~0.4；
(2) after completing step (1), IPTG is added and makes its 50 μM~600 μM of concentration in cultivating system, 18 DEG C~
20 DEG C (such as 18 DEG C or 20 DEG C) culture 6h~18h (such 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 the step (1), the culture can be shaken cultivation.The revolving speed of the shaken cultivation concretely 100rpm~
140rpm more specifically can be 120rpm.
In the step (2), the culture can be shaken cultivation.The revolving speed of the shaken cultivation concretely 100rpm~
140rpm more specifically can be 120rpm.
In the step (2), in described " IPTG is added and makes its 50 μM~600 μM of concentration in cultivating system "
Concentration refers to initial concentration of the IPTG in cultivating system.
In the above method, " the culturing engineering bacterium " may also include step (3): after completing the step (2), be collected by centrifugation
Thallus.Parameter of noncentricity can be centrifuged 10min for 5000rpm.
In the above method, " the culturing engineering bacterium " may also include step (4): the thallus for taking the step (3) to obtain, right
The thallus is crushed, and being then centrifuged for collection supernatant, (supernatant is that the crude protein containing protein UGTCs4 is molten
Liquid).Parameter of noncentricity can be centrifuged 30min for 4 DEG C, 13000rpm.
In the above method, " the culturing engineering bacterium " may also include step (5): the supernatant for taking the step (4) to obtain,
Affinity chromatography purifying is carried out, solution (as protein UGTCs4 solution) after column is obtained.
In the step (5), the affinity chromatography purifying, which specifically includes, first balances nickel column with lysis buffer, then
The supernatant that step described in loading (4) obtains, then successively with the washing buffer of the imidazoles containing 20mmol/L, imidazoles containing 80mmol/L
Washing buffer and the imidazoles containing 100mmol/L washing buffer rinse nickel column, finally with the washing of the imidazoles containing 250mmol/L
Buffer rinses nickel column.
The lysis buffer can be pH7.4,100mmol/L of NaCl containing 300mmol/L and 10mmol/L imidazoles
Tris-HCl buffer.
The washing buffer can be pH7.4, the 100mmol/L Tris-HCl buffer of the NaCl containing 300mmol/L.
In the above method, " the culturing engineering bacterium " may also include step (6): under the conditions of 4 DEG C, by the step (5)
Solution carries out dialysis desalination after obtained column excessively.
In the above method, before carrying out the step (1), can also it carry out following steps (A): by the recombinant bacterium Dan Ke
It is grand to be inoculated into 5mL fluid nutrient medium, 35 DEG C~39 DEG C (such 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 (such as 100rpm~120rpm, 120rpm~140rpm, 100rpm, 120rpm or 140rpm) shaken cultivation
10h~14h (such as 10h~12h, 12h~14h, 10h, 12h or 14h), obtains seed liquor；The seed liquor is inoculated into liquid
Culture medium, inoculum concentration are 1:100 (volume ratio).
Any of the above-described fluid nutrient medium can be the LB liquid medium containing 50 μ g/mL kanamycins.
The protein UGTCs4 being prepared using any of the above-described the method also belongs to protection scope of the present invention.
The application for the protein UGTCs4 being prepared using any of the above-described the method also belongs to protection model of the invention
Enclose, the application of the protein UGTCs4 is following c1) or c2) or c3) c4) or c5) or c6):
C1) the application as crocetin glycosyl transferase；
C2) the application in the product that preparation has crocetin glycosyl transferase function.
C3 the application in glycosylation) occurs in catalysis crocetin；
C4 application in glycosylated product) occurs for being catalyzed crocetin in preparation.
C5) the application in production crocin glycosides 5 and/or crocin glycosides 3；
C6) application in the product for producing crocin glycosides 5 and/or crocin glycosides 3 is being prepared.
In above-mentioned application, " production crocin glycosides 5 and/or the crocin glycosides 3 " is using crocetin as substrate.
It is demonstrated experimentally that protein UGTCs4 can be prepared using the method provided by the present invention for preparing protein UGTCs4,
Protein UGTCs4 has crocetin glycosyl transferase activity, can sequentially glycosylate crocetin and form 5 He of crocin glycosides
Crocin glycosides 3.The method provided by the present invention for preparing protein UGTCs4 is to production crocin glycosides 5 and/or safflower
It is important that there is the product of crocetin glycosyl transferase function or catalysis crocetin generation glycosylation to all have for plain glycosides 3, preparation
Application value.
Detailed description of the invention
Fig. 1 is the pcr amplification product of UGTCs4 gene.
Fig. 2 is the influence of expression of the various concentration IPTG to recombination crocetin glycosyl transferase.
Fig. 3 is the influence of expression of the different induction times to recombination crocetin glycosyl transferase.
Fig. 4 is the influence of expression of the different cell concentrations to recombination crocetin glycosyl transferase.
Fig. 5 is the influence of expression of the different inducing temperatures to recombination crocetin glycosyl transferase.
Fig. 6 is the influence of the expression of different inducing temperatures and induction time to recombination crocetin glycosyl transferase.
Fig. 7 is the SDS-PAGE result using ni-sepharose purification crocetin glycosyl transferase solution.
The experimental result that Fig. 8 is in 2 step 2 of embodiment 1.
The experimental result that Fig. 9 is in 2 step 2 of embodiment 2.
The experimental result that Figure 10 is in 2 step 2 of embodiment 3.
The experimental result that Figure 11 is in 2 step 2 of embodiment 4.
Figure 12 is the mass spectrogram of crocetin.
Figure 13 is the mass spectrogram of crocin glycosides 5.
Specific embodiment
The present invention is further described in detail With reference to embodiment, and the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Carrier pET28a (+) is invitrogen Products；E. coli bl21 (DE3) is Tiangeng biochemical technology (north
Capital) Co., Ltd's product；Nickel column is U.S. GE Products, catalog number 04003272-EE.DTT is Amersco company
Product, catalog number 0281.UDPG (UDP-Glucose) is sigma Products, catalog number U4625.Safranine
Spending acid is Chromedex Products, catalog number ASB00003885-010.C18 reversed-phase column (4.6mm × 250mm) is
Japanese GL Sciences Inc product.High performance liquid chromatograph and diode array detector are Japanese Shimadzu Corporation product.It is super
It is Xin Zhi bio tech ltd, Ningbo product that sound, which is crushed instrument,.
Safflower suspension cell in following embodiments is with reference to Chen Shuan, Wang Xiaodong etc., safflower cell suspension cultures
The foundation of system and optimization biotechnology are notified to 2010. (07): the method preparation in 157-160..
LB liquid medium: being dissolved in 100mL deionized water for 1g NaCl, 0.5g yeast extract and 1g tryptone, adjusts
Saving 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, the 50mM Tris-HCl buffer of beta -mercaptoethanol.
Lysis buffer is pH7.4,100mmol/L Tris-HCl of NaCl containing 300mmol/L and 10mmol/L imidazoles
Buffer.
Washing buffer is pH7.4, the 100mmol/L Tris-HCl buffer of the NaCl containing 300mmol/L.
Protein shown in sequence 2 by sequence table is named as protein UGTCs4, the sequence of encoding gene such as sequence table
Shown in column 1.
Embodiment 1, the preparation for recombinating crocetin glycosyl transferase
One, the building of recombinant plasmid
1, the total serum IgE reverse transcriptase reverse transcription is gone out the by the total serum IgE that safflower suspension cell is extracted using Trizol method
One chain cDNA.
2, artificial synthesized primers F 1:5 '-CCATATG(underscore is in restricted to GAGCAGAAAGATGAGAACGGAA-3 '
Enzyme cutting Nde I identifies sequence) and R1:5 '-CGTCGAC(underscore is restricted to TTTACAACAATGATCAATGAACTCCT-3 '
Restriction endonuclease Sal I identifies sequence).
3, for the cDNA obtained using step 1 as template, the F1 and R1 synthesized using step 2 carries out PCR amplification as primer, obtains about
The pcr amplification product of 1388bp.It is detected with 1% agarose gel electrophoresis, (M is DNA Marker, swimming lane 1 to the result is shown in Figure 1
For pcr amplification product, arrow meaning 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 recycle；72℃10min.
4, the pcr amplification product obtained in I double digestion step 3 of restriction enzyme Nde I and Sal, recycling is about
The segment of 1386bp.
5, with I double digestion carrier pET28a (+) of restriction enzyme Nde I and Sal, the carrier bone of about 5350bp is recycled
Frame.
6, the segment that step 4 obtains is connect with the carrier framework that step 5 obtains, obtains recombinant plasmid pET28a-
UGTCs4。
According to sequencing result, structure is carried out to recombinant plasmid pET28a-UGTCs4 and is described as follows: by carrier pET28a (+)
Nde I to identify that the DNA small fragment between sequence and Sal I identify sequence replaces with nucleotide sequence be in sequence table shown in sequence 1
DNA molecular.In recombinant plasmid pET28a-UGTCs4, on DNA molecular shown in the sequence 1 of sequence table and carrier framework
The coded sequence of His-tag label (being made of 6 histidine residues) merges, and merges base shown in the sequence 3 of formation sequence table
Cause, with the protein UGTCs4 of His-tag label shown in the sequence 4 of expressed sequence table, the albumen with His-tag label
Matter UGTCs4 is to recombinate crocetin glycosyl transferase.The expection molecular weight of protein shown in the sequence 4 of sequence table is
54.8KD。
Two, groping for the optimum condition of the expression of crocetin glycosyl transferase is recombinated
1, influence of the various concentration IPTG to the expression of recombination crocetin glycosyl transferase
1. the recombinant plasmid pET28a-UGTCs4 of step 1 building is imported Escherichia coli Rosetta (DE3), weight is obtained
Group bacterium, is named as Rosetta (DE3)-pET28a-UGTCs4 for the recombinant bacterium.
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins
Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture 1 0.3mL of bacterium solution is taken, with 1:100 (body
Product ratio) it is inoculated in the LB liquid medium that 30mL contains 50 μ g/mL kanamycins, 37 DEG C, 180rpm shaken cultivation to OD600Value 0.3
~0.4, obtain culture bacterium solution 2.
3. after completing step 2., taking culture bacterium solution 2, centrifugation, the thallus before obtaining IPTG induction.
4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system
Degree is 50 μM, 100 μM, 200 μM, 300 μM, 400 μM, 500 μM, 600 μM or 700 μM, then 37 DEG C, 120rpm shaken cultivation
2.5h, thalline were collected by centrifugation, the thallus after obtaining IPTG induction.
5. the thallus after thallus and IPTG induction before taking IPTG to induce respectively, is added LB liquid medium to OD600Value is adjusted
Whole to consistent, 25 DEG C, 9000rpm centrifugation 1min abandon supernatant, collect thallus.
6. the thallus for taking step 5. to obtain, is added sample-loading buffer, boiling water bath boils 13min；Then 25 DEG C, 9000rpm
It is centrifuged 10min, supernatant is collected and carries out SDS-PAGE.
Experimental result is shown in Fig. 2, (M is albumen marker, and 1 is the thallus before IPTG induction, after 2 is 50 μM of IPTG inductions
Thallus, 3 be the thallus after 100 μM of IPTG inductions, and 4 be the thallus after 200 μM of IPTG inductions, after 5 is 300 μM of IPTG inductions
Thallus, 6 be the thallus after 400 μM of IPTG induction, and 7 be the thallus after 500 μM of IPTG inductions, and 8 be 600 μM of IPTG inductions
Thallus afterwards, 9 be the thallus after 700 μM of IPTG inductions, and arrow meaning is target protein).The result shows that recombination crocetin
The best IPTG induced concentration of the expression of glycosyl transferase is 50 μM~600 μM；When IPTG induced concentration is 700 μM, recombination
The expression of crocetin glycosyl transferase has a degree of inhibition.
2, influence of the difference IPTG induction time to the expression of recombination crocetin glycosyl transferase
1. in step 1 1..
2. in step 1 2..
3. in step 1 3..
4. after completing step 2., after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG exists
Concentration in induction system is 200 μM, then 37 DEG C, 120rpm shaken cultivation, incubation time 30min, 1h, 1.5h, 2h,
2.5h, 3h or 4h, thalline were collected by centrifugation, the thallus after obtaining IPTG induction.
5. in step 1 5..
6. in step 1 6..
Experimental result is shown in Fig. 3, (M is albumen marker, and 1 is the thallus before IPTG induction, after 2 induce 30min for IPTG
Thallus, 3 induce the thallus after 1h for IPTG, and 4 induce the thallus after 1.5h for IPTG, and 5 induce the thallus after 2h for IPTG, and 6 are
IPTG induces the thallus after 2.5h, and 7 induce the thallus after 3h for IPTG, and 8 induce the thallus after 4h for IPTG, and arrow meaning is mesh
Mark albumen).The result shows that the best IPTG induction time of the expression of recombination crocetin glycosyl transferase is 30min~4h.
3, influence of the different cell concentrations to the expression of recombination crocetin glycosyl transferase
1. in step 1 1..
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins
Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture 1 0.3mL of bacterium solution is taken, with 1:100 (body
Product ratio) it is inoculated in the LB liquid medium that 30mL contains 50 μ g/mL kanamycins, 37 DEG C, 180rpm shaken cultivation obtain culture bacterium
Liquid 2 cultivates the OD of bacterium solution 2600Value is 0.32,0.41,0.54,0.63 or 0.76.
3. in step 1 3..
4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system
Degree is 200 μM, and then 37 DEG C, 120rpm shaken cultivation 2.5h, thalline were collected by centrifugation, the thallus after obtaining IPTG induction.
5. in step 1 5..
6. in step 1 6..
Experimental result is shown in Fig. 4, (M is albumen marker, and 1 is the thallus before IPTG induction, and 2 be OD600The culture that value is 0.32
Thallus after bacterium solution 2IPTG induction, 3 be OD600Value is the thallus after 0.41 culture bacterium solution 2IPTG induction, and 4 be OD600Value is
Thallus after 0.54 culture bacterium solution 2IPTG induction, 5 be OD600Thallus after the culture bacterium solution 2IPTG induction that value is 0.63,6
For OD600Value is the thallus after 0.76 culture bacterium solution 2IPTG induction, and arrow meaning is target protein).The result shows that recombination hiding
The OD of the best cell concentration of the expression of safflower acid glycosyl transferase600Value is 0.3~0.8.
4, influence of the different inducing temperatures to the expression of recombination crocetin glycosyl transferase
(1) influence of 28 DEG C of the inducing temperature expression to recombination crocetin glycosyl transferase
1. in step 1 1..
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins
Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture 1 0.3mL of bacterium solution is taken, with 1:100 (body
Product ratio) it is inoculated in the LB liquid medium that 30mL contains 50 μ g/mL kanamycins, 28 DEG C, 120rpm shaken cultivation to OD600Value 0.3
~0.4, obtain culture bacterium solution 2.
3. in step 1 3..
4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system
Degree is 200 μM, and then 28 DEG C, 120rpm shaken cultivation, bacterium is collected by centrifugation in incubation time 3h, 4h, 5h, 6h, 7h, 8h or 9h
Body, the thallus after obtaining IPTG induction.
5. in step 1 5..
6. in step 1 6..
Experimental result is shown in Fig. 5, (M is protein low molecular weight marker, and 1 induces for the thallus before IPTG induction, 2 for IPTG
Thallus after 3h, 3 induce the thallus after 4h for IPTG, and 4 induce the thallus after 5h for IPTG, and 5 induce the thallus after 6h for IPTG,
6 induce the thallus after 7h for IPTG, and 7 induce the thallus after 8h for IPTG, and 8 induce the thallus after 9h for IPTG, and arrow meaning is
Target protein).The result shows that recombination crocetin glycosyl transferase is primarily present in inclusion body, it is seen that recombination crocetin sugar
The temperature of the expression of based transferase can not be 28 DEG C.
(2) influence of 37 DEG C of the inducing temperature expression to recombination crocetin glycosyl transferase
According to the method for step (1), 37 DEG C are replaced with by 28 DEG C, other steps are constant, as a result recombinate crocetin sugar
Based transferase is also primarily present in inclusion body, it is seen that the temperature of the expression of recombination crocetin glycosyl transferase can not be 37 DEG C.
(3) influence of 18 DEG C of the inducing temperature expression to recombination crocetin glycosyl transferase
1. in step 1 1..
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins
Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture 1 0.3mL of bacterium solution is taken, with 1:100 (body
Product ratio) 30mL is inoculated in containing 50 μ g/mL kanamycins LB liquid mediums, 18 DEG C, 120rpm shaken cultivation to OD600Value 0.3~
0.4, obtain culture bacterium solution 2.
3. in step 1 3..
4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system
Degree is 200 μM, then 18 DEG C, 120rpm shaken cultivation, incubation time 6h, 8h, 10h, 12h, 14h, 16h or 18h, and centrifugation is received
Collect thallus, the thallus after obtaining IPTG induction.
5. in step 1 5..
6. in step 1 6..
Experimental result is shown in Fig. 6, (M is albumen marker, and 1 is the thallus before IPTG induction, and 2 induce the bacterium after 6h for IPTG
Body, 3 induce the thallus after 8h for IPTG, and 4 induce the thallus after 10h for IPTG, and 5 induce the thallus after 12h for IPTG, and 6 are
IPTG induces the thallus after 14h, and 7 induce the thallus after 16h for IPTG, and 8 induce the thallus after 18h for IPTG, and arrow meaning is
Target protein).The result shows that 18 DEG C, the inducible expression for recombinating crocetin glycosyl transferase of IPTG induction 6h~18h.
Three, the expression and purification of crocetin glycosyl transferase is recombinated
1. recombinant plasmid pET28a-UGTCs4 is imported Escherichia coli Rosetta (DE3), recombinant bacterium is obtained, by the recombination
Bacterium is named as Rosetta (DE3)-pET28a-UGTCs4.
2. the monoclonal of Rosetta (DE3)-pET28a-UGTCs4 is inoculated in the LB that 5mL contains 50 μ g/mL kanamycins
Fluid nutrient medium, 37 DEG C, 120rpm shaken cultivation 12h obtain culture bacterium solution 1.Culture 1 0.3mL of bacterium solution is taken, with 1:100 (body
Product ratio) 30mL is inoculated in containing 50 μ g/mL kanamycins LB liquid mediums, 18 DEG C, 120rpm shaken cultivation to OD600Value 0.3~
0.4, obtain culture bacterium solution 2.
3. after completing step 2., taking culture bacterium solution 2,5000rpm is centrifuged 10min, collects thallus, before obtaining IPTG induction
Thallus.
4. after completing step 2., IPTG being added into culture bacterium solution 2 and obtains induction system, IPTG is dense in induction system
Degree is 200 μM, and then 18 DEG C, 120rpm shaken cultivation 16h, 5000rpm centrifugation 10min collect thallus, after obtaining IPTG induction
Thallus.
5. the thallus after taking IPTG to induce, washed once with lysis buffer.
6. taking into the thallus of step 5., lysis buffer is added with 1:10 (volume ratio), -20 DEG C is put into and freezes, obtain
Freeze bacterium solution.
7. take into step 6. freeze bacterium solution, rapidly thaw after, set on ice on Ultrasonic Cell Disruptor ultrasonication (ultrasound
Wave power 100W, cyclic program are as follows: broken 6s stops 4s, totally 120 circulations), then 4 DEG C, 13000rpm centrifugation 30min are collected
Supernatant, the bacterial cell disruption supernatant after being named as IPTG induction.
According to 5. to 7. the step of, the thallus after IPTG is induced replace with IPTG induction before thallus, obtain IPTG and lure
The bacterial cell disruption supernatant of leading.
8. first balancing nickel column (flow velocity 1mL/min) with the lysis buffer of 5~10 column volumes, then loading step is 7.
Bacterial cell disruption supernatant (flow velocity 1mL/min) after obtained IPTG induction, then successively with 5 column volumes containing 20mmol/L
The washing buffer of imidazoles, 5 column volumes the imidazoles containing 80mmol/L washing buffer and 5 column volumes contain 100mmol/
The washing buffer of L imidazoles rinses nickel column, to remove most of foreign protein, finally with the miaow containing 250mmol/L of 5 column volumes
The washing buffer of azoles rinses nickel column and collected solution after column, as recombination crocetin glycosyl transferase solution.
Bacterial cell disruption supernatant after IPTG is induced, the bacterial cell disruption supernatant before IPTG induction and step 8. in respectively wash
The elution collection liquid of de- step carries out SDS-PAGE.
Experimental result is shown in Fig. 7, (M is protein molecular weight Marker, and 1 is the bacterial cell disruption supernatant before IPTG induction, and 2 are
Bacterial cell disruption supernatant after IPTG induction, the 3 elution collection liquids eluted for the washing buffer of the imidazoles containing 20mmol/L,
The 4 elution collection liquids eluted for the washing buffer of the imidazoles containing 80mmol/L, 5 is slow for the washing of the imidazoles containing 100mmol/L
The elution collection liquid that fliud flushing is eluted, the 6 elution collection liquids eluted for the washing buffer of the imidazoles containing 250mmol/L).
The result shows that recombination crocetin glycosyl transferase solution shows that unimodal molecular weight band, corresponding molecular weight are 54.8KDa, with
It is expected that molecular weight is consistent.
The content of crocetin glycosyl transferase is recombinated in bacterial cell disruption supernatant after 7. IPTG induction that step obtains
Crocetin glycosyl transferase is recombinated in the recombination crocetin glycosyl transferase solution 8. obtained for 0.03 μ g/ μ L, step
Content is 2.13 μ g/ μ L.Recombination crocetin glycosyl transfer in bacterial cell disruption supernatant after 7. IPTG induction that step obtains
The calculation method of the content of enzyme are as follows: the total protein content of liquid-phase system is detected, then by each band in protein electrophoresis
It carries out gray scale scanning and calculates recombination crocetin glycosyl transferase ratio shared in total protein, recombination safflower is calculated
The content of sour glycosyl transferase.Crocetin glycosyl is recombinated in 8. recombination crocetin glycosyl transferase solution that step obtains to turn
The content for moving enzyme is measured according to Bradford method.
Embodiment 2, the glycosyl transferase that recombination crocetin glycosyl transferase under the conditions of differential responses is detected using HPLC
Activity
One, it dialyses
Under the conditions of 4 DEG C, the recombination crocetin glycosyl transferase solution that embodiment 1 obtains is subjected to dialysis desalination, then
It replaces pH7.5, in 5mM Tris-HCl buffer, finally carries out ultrafiltration concentration egg using the super filter tube that molecular weight is 30MW
It is white, obtain the protein sample for the recombination crocetin glycosyl transferase that purity is 98%.The protein sample is subjected to 25 ammonia of N-terminal
The sequencing of base acid residue, the results showed that, in the sequence such as sequence table of 25 amino acid of the albumen n end sequence 4 from N-terminal the 1st to
Shown in 25.
Two, the glycosyl transferase activity of recombination crocetin glycosyl transferase under the conditions of differential responses is detected using HPLC
1, influence of the differential responses time to the glycosyl transferase activity of recombination crocetin glycosyl transferase
(1) preparation 200 μ L recombinate the external enzymatic reaction system of crocetin glycosyl transferase, the system solvent be pH7.5,
50mM Tris-HCl buffer, solute are 1mM DTT, 5mM UDPG (UDP-Glucose), 50 μM of MgCl2, 10 μM of safflowers
The recombination crocetin glycosyl transferase that acid and 16 μ g step 1 obtain.
(2) by step (1) prepare system be placed in 37 DEG C under the conditions of, then respectively react 0min, 30min, 1h, 1.5h,
2h, 2.5h, 3h or 4h.
(3) after completing step (2), 400 μ L hplc grade methanols is added and terminate reaction, then 13000rpm is centrifuged 10min, receives
Collect supernatant and through 0.45 μm of organic membrane filtration, obtains the to be measured of the external enzymatic reaction of recombination crocetin glycosyl transferase
Sample.
(4) sample to be tested and hiding of the external enzymatic reaction for the recombination crocetin glycosyl transferase for taking step (3) to obtain
Safflower acid standard solution (dissolving crocetin to its concentration with methanol is 10 μM), carries out the detection of HPLC-MS.Using being furnished with
The high performance liquid chromatograph and UV-vis detector of C18 reversed-phase column (4.6mm × 150mm) carry out HPLC detection.Mobile phase by
Methanol (A) and water (B) composition, flow velocity 1mL/min use following condition of gradient elution: in 0~20min, first in mobile phase
The volumn concentration of alcohol at the uniform velocity increases to 50% by 20%, and the volumn concentration of water is at the uniform velocity down to 50% by 80%, carries out line
Property gradient elution；In 20~50min, the volumn concentration of methanol at the uniform velocity increases to 70% by 50% in mobile phase, the body of water
Product percentage composition is at the uniform velocity down to 30% by 50%, carries out linear gradient elution；In 50~55min, with by first alcohol and water according to
The mobile phase 1 that the volume ratio of 7:3 is mixed to get is eluted；In 55~60min, eluted with methanol；In 60~65min
Interior, the volumn concentration of methanol is at the uniform velocity down to 20% by 100% in mobile phase, and the volumn concentration of water is at the uniform velocity risen by 0%
To 80%, linear gradient elution is carried out；In 65~70min, with the stream being mixed to get by first alcohol and water according to the volume ratio of 2:8
Dynamic phase 2 is eluted.Detection wavelength is 440nm.(methanol dissolves crocetin to Simultaneous Quantitative Analysis crocetin standard solution
It is 10 μM to its concentration).Mass Spectrometer Method condition are as follows: scanning of the mass spectrum range 100-2000 (m/z)；Carrier gas: high pure nitrogen, carrier gas stream
Speed: 40arb, secondary air speed: 2arb, blowback gas velocity: 1arb, capillary temperature: 275 DEG C, capillary voltage: 100V, spray
Mist voltage: 3.2V.Experiment in triplicate, repeats to set 10 triangular flasks every time.
Recombinate crocetin glycosyl transferase external enzymatic reaction sample to be tested HPLC detection experimental result is shown in Fig. 8
(CK is crocetin standard items, and abscissa is retention time), under the chromatographic condition, the retention time of crocetin is
35min, the retention time of crocin glycosides 5 are 25min.Experimental result is shown in Figure 12, safflowers for the high resolution mass spectrum of crocetin
Experimental result is shown in Figure 13 for the high resolution mass spectrum of plain glycosides 5.The result shows that sugar of the reaction time to recombination crocetin glycosyl transferase
Based transferase activity influence is more significant, and with the extension of reaction time, crocetin concentration and crocin glycosides 3 gradually subtract
Few, crocin glycosides 5 gradually increases.Therefore, there is recombination crocetin glycosyl transferase sequence glycosylation crocetin to be formed
The function of crocin glycosides 3 and crocin glycosides 5, recombination crocetin glycosyl transferase prepared by embodiment 1 have with safranine
Spending acid is the glycosyl transferase activity of substrate.
2, influence of the different pH value to the glycosyl transferase activity of recombination crocetin glycosyl transferase
(1) preparation 200 μ L recombinate the external enzymatic reaction system of crocetin glycosyl transferase, the system solvent be 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 are 1mM DTT, 5mM UDPG (UDP-Glucose), 50 μM of MgCl2、10μM
The recombination crocetin glycosyl transferase that crocetin and 16 μ g step 1 obtain.
(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.
Recombinate crocetin glycosyl transferase external enzymatic reaction sample to be tested HPLC detection experimental result is shown in Fig. 9
(CK is crocetin standard items, and abscissa is retention time), under the chromatographic condition, the retention time of crocetin is
35min.The result shows that different pH value are more significant on the glycosyl transferase activity influence of recombination crocetin glycosyl transferase,
When pH value is 7.5, the glycosyl transferase activity highest of crocetin glycosyl transferase is recombinated.
3, influence of the different metal ions to the glycosyl transferase activity of recombination crocetin glycosyl transferase
(1) preparation 200 μ L recombinate the external enzymatic reaction system of crocetin glycosyl transferase, the system solvent be pH7.5,
50mM Tris-HCl buffer, solute are 1mM DTT, 5mM UDPG (UDP-Glucose), 50 μM of chlorates, 10 μM of safflowers
The recombination crocetin glycosyl transferase that acid and 16 μ g step 1 obtain.Chlorate 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.
Recombinate crocetin glycosyl transferase external enzymatic reaction sample to be tested HPLC detection experimental result is shown in figures
Under the chromatographic condition, the retention time of crocetin is 10 (CK is crocetin standard items, and abscissa is retention time)
35min.The result shows that different metal ions influence more to show on the glycosyl transferase activity of recombination crocetin glycosyl transferase
It writes: Cu2+(with CuCl2Form be added), Zn2+(with ZnCl2Form is added) or Mn2+(with MnCl2Form is added) to recombination safranine
The glycosyl transferase activity of sour glycosyl transferase is spent to have apparent inhibiting effect；Mg2+(with MgCl2Form be added), Ca2+(with Ca
Cl2Form is added) or Na+(being added in the form of NaCl) has the glycosyl transferase activity of recombination crocetin glycosyl transferase bright
Aobvious facilitation can promote to recombinate crocetin glycosyl transferase sequence glycosylation crocetin formation 3 He of crocin glycosides
Crocin glycosides 5.
4, influence of the different concentration of substrate to the glycosyl transferase activity of recombination crocetin glycosyl transferase
(1) preparation 200 μ L recombinate the external enzymatic reaction system of crocetin glycosyl transferase, the system solvent be pH7.5,
50mM Tris-HCl buffer, solute are 1mM DTT, 5mM UDPG (UDP-Glucose), 50 μM of MgCl2, crocetin and
The recombination crocetin glycosyl transferase that 16 μ g step 1 obtain.Concentration of the crocetin in the system is 10 μM, 20 μM, 30
μM, 40 μM, 50 μM or 60 μM.
(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.
Recombinate crocetin glycosyl transferase external enzymatic reaction sample to be tested HPLC detection experimental result is shown in figures
Under the chromatographic condition, the retention time of crocetin is 11 (CK is crocetin standard items, and abscissa is retention time)
35min.The result shows that different concentration of substrate influence more to show on the glycosyl transferase activity of recombination crocetin glycosyl transferase
It writes: when concentration of substrate is 10 μM, recombinating the glycosyl transferase activity highest of crocetin glycosyl transferase；With concentration of substrate
Increase, crocin glycosides 3 constantly accumulates, and crocin glycosides 5 is constantly reduced.
Claims (7)
1. a kind of method for preparing protein UGTCs4, includes the following steps: culturing engineering bacterium, the protein is obtained
UGTCs4；The engineering bacteria is the recombinant bacterium for obtaining recombinant expression carrier importing host strain；The recombinant expression carrier is will
The recombinant plasmid that the encoding gene insertion expression vector of the protein UGTCs4 obtains；
The protein UGTCs4 is following a1) or a2):
A1) amino acid sequence is protein shown in sequence 2 in sequence table；
A2) amino acid sequence is protein shown in sequence 4 in sequence table.
2. the method as described in claim 1, it is characterised in that: the encoding gene of the protein UGTCs4 is following b1) or
B2 DNA molecular shown in):
B1) nucleotide sequence is DNA molecular shown in sequence 1 in sequence table；
B2) nucleotide sequence is DNA molecular shown in sequence 3 in sequence table.
3. the method as described in claim 1, it is characterised in that: the recombinant expression carrier is in the polyclonal position of expression vector
The recombinant plasmid that DNA molecular shown in the sequence 1 of point insetion sequence table obtains.
4. the method as described in claim 1, it is characterised in that: the host strain is Escherichia coli.
5. the method as described in Claims 1-4 is any, it is characterised in that: during " the culturing engineering bacterium ", carry out
IPTG induction.
6. method as claimed in claim 5, it is characterised in that: the method for the IPTG induction is as follows: when cultivating system
OD600nmWhen value is 0.3~0.4, IPTG is added and makes its 50 μM~600 μM of concentration in cultivating system.
7. method as claimed in claim 5, it is characterised in that: " the culturing engineering bacterium " includes the following steps:
(1) it is cultivated 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), IPTG is added and makes its 50 μM~600 μM of concentration in cultivating system, 18 DEG C~20 DEG C
Cultivate 6h~18h.
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