CN107098959B - The preparation method of bumblebee peptidoglycan recognition protein - Google Patents
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Abstract
The present invention relates to molecular biology fields, specifically, it is related to a kind of preparation method of bumblebee peptidoglycan recognition protein, comprising: 1), by the genetic fragment of bumblebee PGRP-SA connect expression vector and convert to Escherichia coli and express, collect the inclusion body protein that expression obtains；2), protein denaturant dissolution will be added after inclusion body protein washing, then carries out renaturation process with renaturation solution；3) it, will be successively purified by flash through molecular sieve column and anion-exchange column after protein concentration.Bumblebee PGRP-SA protein expression condition is optimized in the present invention, it is preferably each during reagent formula, in conjunction with chromatography renaturation is used, method is simple and quick, and operating process is mild, small to egg white injury, obtains the reactive protein of high-purity.
Description
Technical field
The present invention relates to molecular biology fields, in particular to a kind of preparation side of bumblebee peptidoglycan recognition protein
Method.
Background technique
Animal of the insect as most species on the earth, be currently known there are about 1,000,000 kinds, they are lived in mostly is full of
In the adverse circumstances of pathogenic microorganism, insect under pressure is selected to form a set of unique immune defense system in long-term evolution
To fight the invasion of these pathogenic microorganisms.
Insect lacks acquired immunity, only relies on congenital immunity as its defense mechanism.It is micro- for the cause of disease of exotic invasive
For biology, insect is put up a resistance by congenital immunity, and the first step is to identify pathogen-associated molecular mould by pattern recognition receptors
Formula, to activate downstream reaction.The having as pattern recognition receptors in insect: peptidoglycan recognition protein (Peptidoglycan
Recognition Proteins, PGRPs), beta glucan identification albumen (β-Glucan Recognition Proteins,
β-GRP) and Gram-negative bacteria binding protein (Gram-neg-ative Binding Protein, GNBP), but wherein
Most important pattern recognition receptors are peptidoglycan recognition proteins.
PGRPs is initially found in the hemolymph of silkworm, this protein binding bacterium peptide glycan and activating pro-phenoloxidase
Cascade reaction-insect host antimicrobial defenses.PGRP-SA is secreting type PGRPs, identifies gram-positive bacteria or fungal cell
Wall peptide glycan (PGN) activates the expression of Toll signal path induction antibacterial peptide, resists entering for the pathogenic microorganisms such as bacterium and fungi
It invades.Bumblebee gene order-checking discloses bumblebee and possesses 4 PGRPs albumen, the table of its PGRP-SA albumen after bacterium infection stimulation
Up to the significant up-regulation of amount.The peptidoglycan recognition protein PGRP-SA of the insects such as drosophila, diamondback moth and housefly is thin respectively at insect Hi-5
Born of the same parents, Drosophila S 2 cells, Escherichia coli (PET28a+ carrier) are expressed；The patent of diamondback moth PGRP-SA albumen has been applied specially
Benefit simultaneously authorizes (publication number CN103484468A, publication date on January 1st, 2014)；Only has fruit in the research of above-mentioned insect PGRP-SA
After fly PGRP-SA expression, after purified, purity of protein reaches 90% or more.However there is no form insect for the studies above
PGRP-SA albumen prepare with scale and the effective ways of high purity protein purifying.
Bumblebee is effective Pollinating Insect of numerous wild plants and crops, has important economic value and ecological value
Value；Meanwhile threat of the health by multiple pathogenic microorganisms such as bacteriums of bumblebee, important immune molecule in innate immune system
The external preparation of peptidoglycan recognition protein PGRP-SA is there is not yet report, this seriously inhibits bumblebee novel antibacterial immune drugs
Research and application.
In view of this, the present invention is specifically proposed.
Summary of the invention
The object of the present invention is to provide a kind of preparation method of bumblebee peptidoglycan recognition protein, this method can be efficient
Prepare the Active Target albumen that simultaneously renaturation obtains high-purity, high homogeneity.
In order to realize above-mentioned purpose of the invention, the following technical scheme is adopted:
A kind of preparation method of bumblebee peptidoglycan recognition protein, includes the following steps:
1), the genetic fragment of bumblebee PGRP-SA is connected expression vector and converted to Escherichia coli and is expressed, table is collected
The inclusion body protein reached；
2), protein denaturant dissolution will be added after inclusion body protein washing, then carries out renaturation process with renaturation solution；
3) it, will be successively purified by flash through molecular sieve column and anion-exchange column after protein concentration.
During the expression and purification of protein, annealing issues are always a difficult point and hot spot in bioengineering.Its
The formation of critical issue aggregation in renaturation process.When albumen refolding in higher concentrations, the peptide chain of stretching, extension by
In the exposed of hydrophobic grouping, it is more easier the disulfide bond formation aggregation due to hydrophobic interaction or intermolecular mispairing.
Classical refolding method generally uses dilution method and dialysis, and albuminate is directly added into renaturation buffer, high protein by the former
A large amount of precipitatings are often formed under concentration, thus very low protein concentration is required when renaturation；And the latter is cumbersome, is unfavorable for putting
Greatly.Ultrafiltration renaturation can continuously replace denaturant with renaturation buffer, reduce denaturant concentration slowly, and protein concentration
It will not be substantially reduced, and be convenient for automatic operation, but shearing force may cause renaturation good protein is denaturalized again and reduces
Activity yield.There are also pulse renaturation, dropwise addition renaturation and membrane tube streams to add renaturation for other improved classical refolding methods, they cannot
Fundamentally solve the problems, such as aggregate and precipitate.
The chromatography renaturation that the present invention uses is in recent years based on the one kind to grow up on the basis of chromatography principle
New refolding method.This method is milder to albumen, and the protein concentration of processing can be very high, easy to operate, the period is short, quilt
It is widely used in carry out protein renaturation, therefore is developing progressively a kind of combined type purifying side for combining purifying and renaturation
Method.
Compared with prior art, the invention has the benefit that
Bumblebee PGRP-SA protein expression condition is optimized, it is preferably each during reagent formula, in conjunction with
Using chromatography renaturation, method is simple and quick, and operating process is mild, small to egg white injury, obtains the activated protein of high-purity
Matter.
Detailed description of the invention
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art be briefly described, it should be apparent that, it is described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, is also possible to obtain other drawings based on these drawings.
Fig. 1 is the inclusion body protein electrophoretogram collected after bumblebee PGRP-SA (bPGRP-SA) inducing expression；
Fig. 2 is inclusion body protein electrophoretogram of bumblebee PGRP-SA (bPGRP-SA) inclusion body after cleaning solution washs；
Fig. 3 is bumblebee PGRP-SA (bPGRP-SA) Superdex 200Increase purifying figure；
Fig. 4 is bumblebee PGRP-SA (bPGRP-SA) Resource Q purifying figure；
Fig. 5 is bumblebee PGRP-SA (bPGRP-SA) SDS-PAGE qualification figure after purification.
Specific embodiment
The present invention relates to the purifying of the vivoexpression of peptidoglycan recognition protein PGRP-SA a kind of, refolding and multi-step
The foundation of method；It is important in reacting more particularly to wild plant with the important congenital antibacterial immunity of Pollinating Insect-bumblebee of crops
Identify the external preparation method of molecule PGRP-SA albumen.
A kind of preparation method of bumblebee peptidoglycan recognition protein, includes the following steps:
1), the genetic fragment of bumblebee PGRP-SA is connected expression vector and converted to Escherichia coli and is expressed, table is collected
The inclusion body protein reached；
2), protein denaturant dissolution will be added after inclusion body protein washing, then carries out renaturation process with renaturation solution；
3) it, will be successively purified by flash through molecular sieve column and anion-exchange column after protein concentration.
Preferably, preparation method as described above, the genetic fragment of the bumblebee PGRP-SA is as shown in SEQ ID NO:1.
In protein expression, the size of selected segment and electrically charged situation have very big influence to expression efficiency.
Preferably, preparation method as described above, the acquisition methods of the genetic fragment of the bumblebee PGRP-SA are as follows:
Using bumblebee cDNA as template, drawn with downstream shown in upstream primer shown in SEQ ID NO:2 and SEQ ID NO:3
Object is expanded；
Preferably, annealing temperature is 57 DEG C~59 DEG C, and cycle-index is 28~32 times；
In some embodiments, it is expanded when amplification using Takara company LATaq enzyme；
In some embodiments, the condition that PCR reacts when amplification are as follows:
1. 92 DEG C~95 DEG C 3~8min of denaturation；
2. 92 DEG C~95 DEG C 25~35s of denaturation；
3. 57 DEG C~59 DEG C 0.5~2min of annealing；
4. 70 DEG C~73 DEG C 20~40s of extension；
2.~4. 28~32 time it repeats；
5. 70 DEG C~73 DEG C 7~12min of extension；
Optionally, 6. 0~6 DEG C of lasting heat preservation.
It is furthermore preferred that in some embodiments, the condition of PCR reaction when amplification are as follows:
1. 93 DEG C~95 DEG C 3~6min of denaturation；
2. 93 DEG C~95 DEG C 27~32s of denaturation；
3. 57 DEG C~59 DEG C 1~1.5min of annealing；
4. 71 DEG C~73 DEG C 25~35s of extension；
2.~4. 29~31 time it repeats；
5. 71 DEG C~73 DEG C 8~11min of extension；
Optionally, 6. 2~6 DEG C of lasting heat preservations.
Preferably, preparation method as described above, the expression vector are PET21a+ carrier.
Preferably, preparation method as described above washs cleaning solution used when the inclusion body protein in step 2)
Selected from cleaning solution A and/or cleaning solution B；
The ingredient of the cleaning solution A includes 0.4%~0.6% (v/v) Triton-100,45~55mM Tris-Cl, and 280
~320mM NaCl, 8~12mM EDTA-Na2, 8~12mM DTT, pH=7.8~8.2；
The ingredient of the cleaning solution B includes 45~55mM Tris-Cl, 80~120mM NaCl, 8~12mM EDTA-
Na2, 8~12mM DTT, pH=7.8~8.2；
Preferably, the ingredient of the cleaning solution A includes 5% (v/v) Triton-100,47~52mM Tris-Cl, 290~
310mM NaCl, 9~11mM EDTA-Na2, 9~11mM DTT, pH=7.8~8.2；
The ingredient of the cleaning solution B includes 47~52mM Tris-Cl, 90~110mM NaCl, 9~11mM EDTA-
Na2, 9~11mM DTT, pH=7.8~8.2；
Preferably, it is washed 1~2 time with cleaning solution B again after washing 1~3 time with cleaning solution A；
It is furthermore preferred that being washed 1 time with cleaning solution B again after washing 2~3 times with cleaning solution A.
Preferably, preparation method as described above includes the guanidine hydrochloride of 5~7mol in the protein denaturant；
It is furthermore preferred that the ingredient of the protein denaturant includes:
5~7M guanidine hydrochloride, 8%~12% (v/v) glycerol, 45~55mM Tris, 90~110mM NaCl, 8~12mM
EDTA-Na2, 8~12mM DTT, pH=7.8~8.2；
5.5~6.5M guanidine hydrochloride, 9%~11% (v/v) glycerol, 47~53mM Tris, 95~105mM NaCl, 9~
11mM EDTA-Na2, 9~11mM DTT, pH=7.9~8.1.
Preferably, when by the solubilization of inclusion bodies such as protein denaturant, the concentration of inclusion body is adjusted to 25~35mg/
ml；Too high concentration aggressiveness precipitating easy to form, too low concentration are uneconomical.
Preferably, the ingredient of preparation method as described above, the renaturation solution includes:
80~110mM Tris, 380~420mM L-Arg HCl (arginine hydrochloric acid), 1.8~2.2mM EDTA, 4.5~
5.5mM GSH (reduced glutathione), 0.4~0.6mM GSSG (oxidized form of glutathione), pH=7.8~8.2；
It is furthermore preferred that the ingredient of the renaturation solution includes:
90~105mM Tris, 390~410mM L-Arg HCl, 1.9~2.1mM EDTA, 4.8~5.2mM GSH,
0.4~0.6mM GSSG, pH=7.9~8.1.
The present invention carries out renaturation using the method for oxidation-reduction pair (redox), comes by adjusting the ratio of GSSG/GSH
Control more accurate redox potential.
Preferably, preparation method as described above, the condition that renaturation process is carried out with renaturation solution are as follows:
In 16~20mg inclusion body protein: inclusion body protein is added renaturation solution and stirs renaturation by the ratio of 100ml renaturation solution
10h~14h；
It is furthermore preferred that inclusion body protein is added dropwise when renaturation solution is added；
It is furthermore preferred that the rate of stirring is 1~3 turn/s, rotor length 3.0cm, stirred in 2 DEG C~6 DEG C.
Preferably, preparation method as described above, in step 3), the concentration is that 500ml is contained inclusion body protein
Renaturation solution be concentrated into 3.5~4.5ml.
Preferably, preparation method as described above, the molecular sieve column are Superdex 200Increase；
It more selects, the condition of the molecular sieve column elution are as follows:
Room temperature, the ingredient of eluent include 18~22mM Tris, 45~55mM NaCl, pH=7.8~8.2, with 0.8~
The flow velocity of 1.2ml/min continuously elutes；
It is furthermore preferred that the ingredient of eluent include 19~21mM Tris, 47~52mM NaCl, pH=7.9~8.1, with
The flow velocity of 0.9~1.1ml/min continuously elutes.
Preferably, preparation method as described above, the anion-exchange column are Resource Q；
It is furthermore preferred that the combination liquid of the anion-exchange column is 8~12mM Tris, 8~12mM NaCl, pH=7.8
~8.2；
It is furthermore preferred that the combination liquid of the anion-exchange column is 9~11mM Tris, 9~11mM NaCl, pH=7.9
~8.1；
Preferably, the condition of the anion-exchange column elution are as follows:
Room temperature, the ingredient of eluent include 8~12mM Tris, 180~220mM NaCl, pH=7.8~8.2, continuous ladder
Degree elutes, and the eluent of 45%~55% concentration is continuously flowed into 40~60min；
It is furthermore preferred that the ingredient of eluent includes 9~11mM Tris, 190~210mM NaCl, pH=7.9~8.1 connect
Continue gradient elution, the eluent of 47%~53% concentration is continuously flowed into 45~55min.
The charge that ion-exchange chromatography can use protein is adsorbed, and the denaturant concentration by changing eluant, eluent makes
Its renaturation, not only can with purifying protein, also can further progress renaturation, be effectively prevented the aggregation of protein.
Embodiment of the present invention is described in detail below in conjunction with embodiment, but those skilled in the art will
Understand, the following example is merely to illustrate the present invention, and is not construed as limiting the scope of the invention.It is not specified in embodiment specific
Condition person carries out according to conventional conditions or manufacturer's recommended conditions.Reagents or instruments used without specified manufacturer is
The conventional products that can be obtained by commercially available purchase.
Embodiment 1
The sequence of albumen expressed by the present embodiment is prepared by the following:
The total serum IgE of red as fire bumblebee is extracted by conventional method, is then carried out reverse transcription and is obtained cDNA, expands by template of cDNA
Increase bumblebee PGRP-SA gene, the genetic fragment size about 500bp, sequence is as shown in SEQ ID NO:1.
Upstream primer used is as shown in SEQ ID NO:2 when amplification, and downstream primer is as shown in SEQ ID NO:3.
It is expanded using Takara company LATaq enzyme, amplification condition are as follows:
1. 95 DEG C of denaturation 5min；
2. 95 DEG C of denaturation 30s；
3. 58 DEG C of annealing 1min；
4. 72 DEG C of extension 30s；
2.~4. 30 time it repeats；
5. 72 DEG C of extension 10min；
6. 4 DEG C of lasting heat preservations.
Said gene segment is connected into PET21a+ carrier (restriction endonuclease EcoRI/XhoI), converts colibacillus engineering
Transetta(DE3).Picking monoclonal colonies, 37 DEG C of 200rpm are used as mother liquor after cultivating 12 hours, carry out later a large amount of thin
Bacterium culture simultaneous processing makees a large amount of inducing expressions that the IPTG that concentration is 1mg/ml carries out albumen；Through 4 DEG C, 8000 × g high speed from
Heart 3min collects sediment, ultrasonication (output power 360W, interval time 6sec, working time 12sec, work times 99
It is secondary) inclusion body protein in precipitating is collected afterwards, the electrophoresis result of inclusion body protein is as shown in Figure 1.
It will collect after obtained inclusion body protein washs 2 times with cleaning solution A and washed 1 time, altogether 3 times with cleaning solution B again.
The formula of the cleaning solution A are as follows:
5% (v/v) Triton-100,50mM Tris-Cl, 300mM NaCl, 10mM EDTA-Na2, 10mM DTT, pH
=8.0；
The formula of the cleaning solution B are as follows:
50mM Tris-Cl, 100mM NaCl, 10mM EDTA-Na2, 10mM DTT, pH=8.0；
SDS-PAGE identifies the collecting amount and purity (Fig. 2) of inclusion body, is then added with the concentration calculation of 30mg/ml
6mol guanidine hydrochloride (6M Gua-HCl, 10% glycerol, 50mM Tris pH8.0,100mM NaCl, 10mM EDTA-Na2,
10mM DTT) dissolution.
Configure 500ml renaturation solution:
100mM Tris, 400mM L-Arg HCl, 2.0mM EDTA, 5.0mM GSH, 0.5mM GSSG, pH=8.0.
The inclusion body protein of 90mg is added into renaturation solution dropwise, stirs renaturation, stirring rate: 2 turns/s, rotor length is
3.0cm is stirred 12 hours in 4 DEG C.
Solution 500ml after renaturation is carried out by 4 DEG C of refrigerators, pressure≤0.4M Pa dilutes after being concentrated under reduced pressure, then at 4
DEG C, 3500 × g centrifugal concentrating, protein concentrate is in 4ml.
It is first to purify (Fig. 3) according to through molecular sieve column Superdex 200Increase with molecular weight；
The condition of molecular sieve column elution are as follows:
Room temperature, the ingredient of eluent are as follows:
20mM Tris, 40mM NaCl, pH=8.0, are continuously eluted with the flow velocity of 1.0ml/min；
Albumen after purification is collected, is then according to again through anion-exchange column Resource with the electrically charged amount of protein surface
Q purifies (Fig. 4):
The combination liquid of the anion-exchange column is 10mM Tris, 10mM NaCl, pH=8.0；
The condition of the anion-exchange column elution are as follows:
Room temperature, the ingredient of eluent include 10mM Tris, 200mM NaCl, pH=8.0, continuous gradient elution, 50min
Inside continuously flow into the eluent of 50% concentration.
For albumen after anion-exchange column is eluted using SDS-PAGE identification (Fig. 5), purity of protein reaches 90% or more.
Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, but those skilled in the art should understand that: its
It is still possible to modify the technical solutions described in the foregoing embodiments, or to some or all of the technical features
It is equivalently replaced；And these are modified or replaceed, various embodiments of the present invention skill that it does not separate the essence of the corresponding technical solution
The range of art scheme.
SEQUENCE LISTING
<110>China Agriculture Industitute Bee Research Center
<120>preparation method of bumblebee peptidoglycan recognition protein
<160> 3
<170> PatentIn version 3.3
<210> 1
<211> 519
<212> DNA
<213>artificial sequence
<400> 1
gaattcaacg aaagttgccc tcagattatc ggacggagcg agtggactga tgtagacgca 60
aaaagcataa attatttaat tattcccata ccatatgtca ttattcatca tacagttacc 120
gcagaatgta atactagaag tgaatgtatt gctcaagccg agaacattcg atcttatcac 180
atggatagta atggatggga tgatatagga tactcatttc taatcggtgg agatgggaat 240
gtatatgagg gccgcggctg gaatcgtgaa ggagctcata ccattggata taataaaaaa 300
tctgtcggaa ttggttttat aggaaatttt caagaaaagg ccgcaagtga taaaatgctg 360
aatgccgcgc atgcattaat tcattgtggc aaatcaaaag gaatacttag agaagatatt 420
cgtgtaatcg gtgctaaaca agttacagcc actatgagtc ctggatcaaa acttcagaag 480
caaattaaaa attggcttga atgggttcct actccgtaa 519
<210> 2
<211> 32
<212> DNA
<213>artificial sequence
<400> 2
cggaattcaa cgaaagttgc cctcagatta tc 32
<210> 3
<211> 32
<212> DNA
<213>artificial sequence
<400> 3
ccctcgagtt acggagtagg aacccattca ag 32
Claims (15)
1. a kind of preparation method of bumblebee peptidoglycan recognition protein, which comprises the steps of:
1), the genetic fragment of bumblebee PGRP-SA is connected expression vector and converted to Escherichia coli and is expressed, collection is expressed
The inclusion body protein arrived；
2), protein denaturant dissolution will be added after inclusion body protein washing, then carries out renaturation process with renaturation solution；
3) it, will be successively purified by flash through molecular sieve column and anion-exchange column after protein concentration；
The genetic fragment of the bumblebee PGRP-SA is as shown in SEQ ID NO:1.
2. preparation method according to claim 1, which is characterized in that the acquisition of the genetic fragment of the bumblebee PGRP-SA
Method are as follows:
Using bumblebee cDNA as template, with downstream primer shown in upstream primer shown in SEQ ID NO:2 and SEQ ID NO:3 into
Row amplification；
Annealing temperature is 57 DEG C~59 DEG C, and cycle-index is 28~32 times.
3. preparation method according to claim 1, which is characterized in that the expression vector is PET21a+ carrier.
4. preparation method according to claim 1, which is characterized in that in step 2), when washing the inclusion body protein
Cleaning solution used is selected from cleaning solution A and/or cleaning solution B；
The ingredient of the cleaning solution A includes 0.4%~0.6% (v/v) Triton-100,45~55mM Tris-Cl, 280~
320mM NaCl, 8~12mM EDTA-Na2, 8~12mM DTT, pH=7.8~8.2；
The ingredient of the cleaning solution B includes 45~55mM Tris-Cl, 80~120mM NaCl, 8~12mM EDTA-Na2, 8~
12mM DTT, pH=7.8~8.2.
5. the preparation method according to claim 4, which is characterized in that use cleaning solution B after washing 1~3 time with cleaning solution A again
Washing 1~2 time.
6. preparation method according to claim 1, which is characterized in that include the salt of 5~7mol in the protein denaturant
Sour guanidine.
7. preparation method according to claim 6, which is characterized in that the ingredient of the protein denaturant includes:
5~7M guanidine hydrochloride, 8%~12% (v/v) glycerol, 45~55mM Tris, 90~110mM NaCl, 8~12mM EDTA-
Na2, 8~12mM DTT, pH=7.8~8.2.
8. preparation method according to claim 1, which is characterized in that the ingredient of the renaturation solution includes:
80~110mM Tris, 380~420mM L-Arg HCl, 1.8~2.2mM EDTA, 4.5~5.5mM GSH, 0.4~
0.6mM GSSG, pH=7.8~8.2.
9. preparation method according to claim 8, which is characterized in that the condition for carrying out renaturation process with renaturation solution
Are as follows:
In 16~20mg inclusion body protein: the ratio of 100ml renaturation solution by inclusion body protein be added renaturation solution stirring renaturation 10h~
14h。
10. preparation method according to claim 1, which is characterized in that in step 3), the concentration is to contain 500ml
There is the renaturation solution of inclusion body protein to be concentrated into 3.5~4.5ml.
11. preparation method according to claim 1, which is characterized in that the molecular sieve column is Superdex 200
Increase。
12. preparation method according to claim 11, which is characterized in that the condition of the molecular sieve column elution are as follows:
Room temperature, the ingredient of eluent include 18~22mM Tris, 45~55mM NaCl, pH=7.8~8.2, with 0.8~
The flow velocity of 1.2ml/min continuously elutes.
13. preparation method according to claim 1, which is characterized in that the anion-exchange column is Resource Q.
14. preparation method according to claim 13, it is characterised in that the combination liquid of the anion-exchange column be 8~
12mM Tris, 8~12mM NaCl, pH=7.8~8.2.
15. preparation method according to claim 13, which is characterized in that the condition of the anion-exchange column elution are as follows:
Room temperature, the ingredient of eluent include 8~12mM Tris, 180~220mM NaCl, pH=7.8~8.2, and continuous gradient is washed
It is de-, the eluent of 45%~55% concentration is continuously flowed into 40~60min.
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