CN109207442A - A method of preparing LPOR albumin crystal - Google Patents
A method of preparing LPOR albumin crystal Download PDFInfo
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- CN109207442A CN109207442A CN201811021496.5A CN201811021496A CN109207442A CN 109207442 A CN109207442 A CN 109207442A CN 201811021496 A CN201811021496 A CN 201811021496A CN 109207442 A CN109207442 A CN 109207442A
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Abstract
The invention discloses a kind of methods for preparing LPOR albumin crystal, specifically include LPOR protein expression, LPOR protein purification, TEV digestion, Ni affinity chromatography, molecular sieve and crystallization process.The present invention provides a kind of methods for crystallizing LPOR albumen, to achieve the purpose that the structure to LPOR albumen parses, the structure elucidation for other photaesthesia hypervelocity albumen is laid a good foundation；Simultaneously to thoroughly illustrating and determine that the catalytic mechanism or be engineered of photaesthesia hypervelocity albumen lays a good foundation.
Description
Technical field
The present invention relates to protein engineering fields, more particularly to a kind of method for preparing LPOR albumin crystal.
Background technique
LPOR (light-dependent protochlorophyllide oxidoreductase) is cyanobacteria, algae
With the key enzyme of metaphyte Chlorophyll synthesis.In photosynthesis approach, organism is catalyzed protochlorophyllide Pchlide
Also original 2 kinds of different reductases: one is rely on light LPOR (light-dependent NADPH:
protochlorophyllide oxidoreductase)；Another kind is the Pchlide oxidoreducing enzyme DPOR for not depending on light
(light-independent Pchlide reductase).LPOR or DPOR is as protochlorophyllide reduction catalysts enzyme
Phototrophy biology Determination of Chlorophyll synthesis key enzyme, the two enzymes be widely present in phototrophy biology in, still, DPOR with
LPOR molecular structure, subunit composition, genome encoding and in terms of completely it is different.DPOR is green by leaf
Body genome encoding is made of 3 protein subunits；LPOR is encoded by nuclear genome；The wherein function of DPOR and LPOR
Can be similar, it can be catalyzed the reduction of Pchlide D ring double bond.Wherein, anaerobic photosynthetic bacteria only has DPOR；Gymnosperm, algae
Class, cyanobacteria and photosynthetic bacteria have DPOR and LPOR；Angiosperm only has LPOR.Last decade, regulation, function in LPOR
Research with catalyst mechanism etc. makes great progress, but the mystery due to never opening LPOR three-dimensional structure
Veil to the catalytic mechanism for thoroughly illustrating and determining LPOR or is engineered the barrier that constitutes one kind and can not go beyond, for many years
Come, scientists attempt to be given a clue with the means of computer simulation for biochemist, however these all only speculate, and right
The difficult point of LPOR protein structure parsing, then be purifying and the crystallization process of LPOR albumen.
Protein purification technique is widely used, and based on the structure of protein and function, life is recognized from molecular level
Phenomenon is ordered, the Main way of modern biology development is had become, and studies protein, first have to obtain highly purified and has
The target substance of bioactivity.The preparation work of protein is related to the various aspects knowledge such as physics, chemistry and biology, but basic principle
Nothing more than two aspects: first is that they, which are assigned to, mechanically to separate with the difference of component apportionment ratios several in mixture
Two or several object phases in, such as saltout, organic solvent extract, chromatography and crystallization etc.；Second is that mixture is placed in single object phase
In, so that each component is allocated in same area by the effect in the physics field of force and reach separation purpose, such as electrophoresis, ultracentrifugation surpasses
Filter etc..The integrality that must be noted that preservation large biological molecule in the application of all these methods, prevents acid, alkali, high temperature, acutely
Mechanism and the forfeiture for leading to mentioned substance bioactivity.Although the purification technique of protein is highly developed, right
In the unstable photosensitive protein of some extremes, or the difficult point of protein purification research at present.
Therefore it provides a kind of method for preparing LPOR albumin crystal, parses the structure of LPOR albumen to reach
Purpose the problem of being those skilled in the art's urgent need to resolve.
Summary of the invention
In view of this, the present invention provides a kind of methods for preparing LPOR albumin crystal, so that LPOR albumin crystal is obtained,
Achieve the purpose that the structure to LPOR albumen parses, the structure elucidation for other photaesthesia hypervelocity albumen has established base
Plinth；Simultaneously to thoroughly illustrating and determine that the catalytic mechanism or be engineered of photaesthesia hypervelocity albumen lays a good foundation.
To achieve the goals above, the present invention adopts the following technical scheme:
A method of preparing LPOR albumin crystal, which comprises the following steps:
(1) LPOR protein expression
Plasmid pEBSRC-TEV-LPOR is transferred in host cell, picking monoclonal is screened and is inoculated with, and 3-6h is cultivated
Afterwards, IPTG overnight induction is added, rear thalline were collected by centrifugation, and utilizes I suspension thalline of buffer；
(2) LPOR protein purification
Thallus suspension liquid obtained in step (1) is crushed, supernatant is obtained after centrifugation, and supernatant is affine through Ni
Chromatographic column, and purification column is balanced with buffer I, purification column is rinsed with buffer II, buffer III and buffer IV respectively later,
And the protein peak for eluting each is collected respectively, to be verified to SDS-PAGE；Wherein LPOR is eluted by buffer III, is received
Collect buffer III and elutes albumen；
(3) TEV digestion
Albumen obtained in step (2) is subjected to digestion and removes His label, and examines digesting efficiency using SDS-PAGE；
(4) Ni affinity chromatography
Protein liquid after digestion obtained in step (3) is carried out to be centrifuged off precipitating, carries out loading after filtering, and collect
Albumen in flowing through；
(5) molecular sieve
Loading after albumen obtained in step (4) is concentrated is rushed column using GF buffer, and is collected respectively each
A protein peak, to be verified to SDS-PAGE；
(6) protein concentration crystallizes
Albumen obtained in rapid (5) is concentrated by ultrafiltration, obtains the albumen that concentration is 30mg/mL, and utilize SDS-
The purity and concentration of PAGE verifying gained albumen, form by 18%PEG20000 and 0.1MNaAc, the crystallization of pH4.5 is slow later
In fliud flushing, manipulator is taken to screen, it is brilliant to carry out a large amount of points.
Wherein, the host cell in step (1) of the present invention is C43, without resistant；Used medium is in experimentation
LB。
Wherein, monoclonal carries out screening in step (1) of the present invention and seeded process is preferred are as follows: by plasmid pEBSRC-TEV-
LPOR is transformed into C43 host cell, and is inverted plate, 37 DEG C of overnight incubations；Monoclonal on picking conversion plate is connected to containing anti-
In the small triangular flask of raw element, 37 DEG C, 220r/min overnight incubation is forwarded in second day with 1% inoculum concentration antibiotic big
In triangular flask, 37 DEG C, 220r/min continues to cultivate.
Preferably, centrifugal condition is 4000-6000rmin in step (1) of the present invention, is centrifuged 20min.
Preferably, centrifugal condition is 13000-16000r/min in step (2) of the present invention, is centrifuged 60min.
Preferably, centrifugal condition is 10000-12000r/min in step (4) of the present invention, is centrifuged 15min；Filter method is
Utilize 0.22 μM of membrane filtration.
Disclosed by the invention crystal method may make that crystal diffraction rate is up to obtained in it
Further, plasmid pEBSRC-TEV-LPOR described in step (1) is the plasmid with Amp resistance.
Further, the final concentration of 0.4-1.0mM of IPTG described in step (1).
Further, it is that cryogenic high pressure is broken, temperature 4 that thallus suspension liquid described in step (2), which carries out broken method,
DEG C, pressure 800-1000MPa is crushed three times.
Further, upper quadrat method described in step (5) is loading in batches, each applied sample amount 250-500mL.
Further, it is 0.5-1.2mL/min that GF buffer described in step (5), which rushes the flow velocity of column,.
It can be seen via above technical scheme that compared with prior art, preparing LPOR albumen crystalline substance the present invention provides a kind of
The method of body has following technological merit:
(1) the present invention provides a kind of methods for preparing LPOR albumin crystal, solve important in current photosynthesis
The structure elucidation problem of LPOR albumen has established base thoroughly to illustrate and determining the catalytic mechanism of LPOR albumen or be engineered
Plinth；
(2) method provided by the invention for preparing LPOR albumin crystal, purifying and knot for other photaesthesia hypervelocity albumen
Crystalline substance, and further structure elucidation is laid a good foundation.
Detailed description of the invention
In order to more clearly explain the embodiment of the invention or the technical proposal in the existing technology, to embodiment or will show below
There is attached drawing needed in technical description to be briefly described, it should be apparent that, the accompanying drawings in the following description is only this
The embodiment of invention for those of ordinary skill in the art without creative efforts, can also basis
The attached drawing of offer obtains other attached drawings.
Fig. 1 attached drawing is LPOR protein purification flow chart provided by the invention；
Fig. 2 attached drawing is the peak shape figure of LPOR protein molecular provided by the invention sieve after purification；
Fig. 3 attached drawing is SDS-PAGE electrophoresis result figure after LPOR protein purification provided by the invention；
Fig. 4 attached drawing is the measurement chart of LPOR protein active provided by the invention；
Fig. 5 attached drawing is the albumin crystal figure provided by the invention tentatively obtained；
Fig. 6 attached drawing is primary dcreening operation albumin crystal diffraction point diagram provided by the invention；
Fig. 7 attached drawing is the albumin crystal figure of optimization provided by the invention；
Fig. 8 attached drawing is optimization albumin crystal diffraction point diagram provided by the invention.
Specific embodiment
Following will be combined with the drawings in the embodiments of the present invention, and technical solution in the embodiment of the present invention carries out clear, complete
Site preparation description, it is clear that described embodiments are only a part of the embodiments of the present invention, instead of all the embodiments.It is based on
Embodiment in the present invention, it is obtained by those of ordinary skill in the art without making creative efforts every other
Embodiment shall fall within the protection scope of the present invention.
Embodiment 1
LPOR protein expression.
Plasmid pEBSRC-TEV-LPOR is converted in C43 host cell, plasmid pEBSRC-TEV-LPOR is anti-with Amp
Property, host C43 is without resistance.Used medium is LB in experimentation, is inverted plate, 37 DEG C of overnight incubations.Picking conversion is flat
Monoclonal on plate is connected in the 100mL triangular flask of the Amp containing 100mg/L, 37 DEG C, 220r/min overnight incubation, in second day with
1% inoculum concentration is forwarded in the big triangular flask of antibiotic 1L, and 37 DEG C, 220r/min continues to cultivate.After cultivating 4h, addition is dense eventually
Degree is the IPTG overnight induction of 0.4mM.6000r/min centrifugation 20min receives bacterium after terminating induction, discards supernatant, thallus is used
Buffer I suspends.
Embodiment 2
The purifying of LPOR albumen
As shown in Figure 1, the specific steps of the purifying of LPOR albumen are as follows: it is broken that thallus suspension liquid first passes around cryogenic high pressure cell
Broken machine is crushed, and broken condition is 4 DEG C of temperature, pressure 1000MPa, is crushed three times.Broken liquid is by 16000r/min high speed
It is centrifuged 60min and removes the cell fragment in precipitating, supernatant is the crude enzyme liquid for including destination protein.
Crude enzyme liquid is flowed through into Ni affinity column, destination protein is adsorbed on Ni column, then is balanced and is purified with buffer I
Column rinses purification column, and the protein peak that each is eluted with buffer II, buffer III and buffer IV respectively later
It collects respectively, to verify to SDS-PAGE, wherein LPOR is eluted by buffer III, is collected buffer III and is eluted albumen.
TEV digestion
Obtained albumen 4 DEG C of progress enzymes in dialysis buffer are stayed overnight, and His label is removed in excision, and is examined using SDS-PAGE
Test digesting efficiency.
Ni affinity chromatography
Protein liquid has many precipitatings after digestion overnight, and 11000r/min is centrifuged 15min removal precipitating, and through 0.22 μM of filter membrane
Loading after filtering.The destination protein for removing His label largely flows out in flowing through, and collection flows through middle albumen, further increases
Purity of protein.
Molecular sieve
Destination protein obtained by secondary Ni column is concentrated, in batches loading, each applied sample amount 250-500mL；Utilize GF buffer
Rush column, flow velocity 1mL/min；Each protein peak is collected respectively, as a result as shown in Fig. 2, molecular sieve obtains two as shown in Figure 2
Obvious protein peak.Each protein peak being collected into is subjected to SDS-PAGE verifying, as a result as shown in figure 3, wherein the in Fig. 3
One swimming lane is Marker, and the second swimming lane is the first peak in Fig. 2, and third swimming lane is the second peak in Fig. 2；As shown in Figure 3 second
First peak that swimming lane, that is, molecular sieve obtains is foreign protein, and the albumen that third swimming lane, that is, second, molecular sieve peak obtains is target egg
It is white, although 3 band of the swimming lane of third as the result is shown of SDS-PAGE, molecular sieve illustrate the result is that an apparent protein peak
Part albumen obtained in this peak has phenomenon of rupture, and two small albumen of molecular weight below add up target exactly above
Molecular weight of albumen.
Embodiment 3
The crystallization of LPOR albumen
Using Amicon Ultra-30k ultrafiltration membrane protein concentrate, and measuring concentration using Nanodrop is 30mg/mL, is taken
After 1 μ L protein concentrate carries out SDS-PAGE verifying purity and concentration, conventional crystallization buffer (2- methyl -2,4- penta 2 is utilized
Alcohol) starting point crystalline substance, it takes manipulator to screen, to the crystallization condition of long crystal, it is brilliant to carry out a large amount of points；
Determination of activity is carried out to obtained LPOR albumin crystal: taking black to be protected from light 96 orifice plates, under being separately added into A1-A
Column substance: A1 water 50ul；A2NADPH 0.5ul, water 49.5ul；A3Pchlid 5ul, water 45ul；A4 albumin crystal 3-4,
Pchlid 5ul, NADPH 0.5ul, water 44.5ul.96 orifice plates are put into nucleic acid-protein detection system, exciting light 450nm is set,
5s emits light 700nm, detects the peak value of 700nm.Obtained LPOR albumin crystal carries out determination of activity result such as Fig. 4, by Fig. 4
It is found that there is proteinase activity under the conditions of obtained LPOR albumin crystal is existing for the Pchlide and NADPH.It will wherein obtain
LPOR albumin crystal carries out the preliminary diffraction of X-ray, as a result such as Fig. 5-6.Meanwhile the present invention continues to optimize the crystalline of LPOR
Amount, is screened through a large amount of crystallization condition, and final determination is formed by 18%PEG20000 and 0.1MNaAc, and the crystallization of pH4.5 is slow
In fliud flushing, manipulator is taken to screen, to the crystallization condition of long crystal, carries out a large amount of point crystalline substances, the crystal of better quality can be obtained, such as
Fig. 7-8, i.e., after the crystalline quality of optimization LPOR, crystal diffraction rate is reachable
The present invention discloses a kind of method for preparing LPOR albumin crystal, parses to reach to the structure of LPOR albumen
Purpose, for other photaesthesia exceed the speed limit albumen structure elucidation lay a good foundation；Illustrate and determine that photaesthesia is super to thorough simultaneously
The catalytic mechanism of fast albumen or be engineered is laid a good foundation.
Each embodiment in this specification is described in a progressive manner, the highlights of each of the examples are with other
The difference of embodiment, the same or similar parts in each embodiment may refer to each other.For device disclosed in embodiment
For, since it is corresponded to the methods disclosed in the examples, so being described relatively simple, related place is said referring to method part
It is bright.
The foregoing description of the disclosed embodiments enables those skilled in the art to implement or use the present invention.
Various modifications to these embodiments will be readily apparent to those skilled in the art, as defined herein
General Principle can be realized in other embodiments without departing from the spirit or scope of the present invention.Therefore, of the invention
It is not intended to be limited to the embodiments shown herein, and is to fit to and the principles and novel features disclosed herein phase one
The widest scope of cause.
Claims (6)
1. a kind of method for preparing LPOR albumin crystal, which comprises the following steps:
(1) LPOR protein expression
Plasmid pEBSRC-TEV-LPOR is transferred in host cell, picking monoclonal is screened and is inoculated with, after cultivating 3-6h,
IPTG overnight induction is added, rear thalline were collected by centrifugation, and utilizes I suspension thalline of buffer；
(2) LPOR protein purification
Thallus suspension liquid obtained in step (1) is crushed, supernatant is obtained after centrifugation, and by supernatant through Ni affinity chromatography
Column, and purification column is balanced with buffer I, purification column is rinsed with buffer II, buffer III and buffer IV respectively later, and will
Each protein peak eluted is collected respectively, to verify to SDS-PAGE；Wherein LPOR is eluted by buffer III, is collected
Buffer III elutes albumen；
(3) TEV digestion
Albumen obtained in step (2) is subjected to digestion and removes His label, and examines digesting efficiency using SDS-PAGE；
(4) Ni affinity chromatography
Protein liquid after digestion obtained in step (3) is carried out to be centrifuged off precipitating, loading is carried out after filtering, and collect and flow through
In albumen；
(5) molecular sieve
Loading after albumen obtained in step (4) is concentrated rushes column using GF buffer, and collects each egg respectively
White peak, to be verified to SDS-PAGE；
(6) protein concentration crystallizes
Albumen obtained in rapid (5) is concentrated by ultrafiltration, concentration is obtained and is the albumen of 30mg/mL, and tested using SDS-PAGE
The purity and concentration of card gained albumen, form by 18%PEG20000 and 0.1MNaAc, the crystallization buffer of pH4.5 later
In, it takes manipulator to screen, it is brilliant to carry out a large amount of points.
2. a kind of method for preparing LPOR albumin crystal according to claim 1, which is characterized in that described in step (1)
Plasmid pEBSRC-TEV-LPOR is the plasmid with Amp resistance.
3. a kind of method for preparing LPOR albumin crystal according to claim 1, which is characterized in that described in step (1)
The final concentration of 0.4-1.0mM of IPTG.
4. a kind of method for preparing LPOR albumin crystal according to claim 1, which is characterized in that described in step (2)
Thallus suspension liquid carries out broken method, and cryogenic high pressure is broken, and temperature is 4 DEG C, pressure 800-1000MPa, is crushed three times.
5. a kind of method for preparing LPOR albumin crystal according to claim 1, which is characterized in that described in step (5)
Upper quadrat method is loading in batches, each applied sample amount 250-500mL.
6. a kind of method for preparing LPOR albumin crystal according to claim 1, which is characterized in that described in step (5)
The flow velocity that GF buffer rushes column is 0.5-1.2mL/min.
Priority Applications (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811021496.5A CN109207442A (en) | 2018-09-03 | 2018-09-03 | A method of preparing LPOR albumin crystal |
| PCT/CN2019/103936 WO2020048412A1 (en) | 2018-09-03 | 2019-09-02 | Method for purifying and crystallizing lpor protein and use thereof |
| US16/993,847 US11001813B2 (en) | 2018-09-03 | 2020-08-14 | Method for purificating and crystallizating LPOR protein and use thereof |
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| CN201811021496.5A CN109207442A (en) | 2018-09-03 | 2018-09-03 | A method of preparing LPOR albumin crystal |
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| CN201811021496.5A Pending CN109207442A (en) | 2018-09-03 | 2018-09-03 | A method of preparing LPOR albumin crystal |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111675754A (en) * | 2020-05-19 | 2020-09-18 | 四川一埃科技有限公司 | Botulinum toxin protein crystal structure, crystal preparation method and resolution method |
| CN114507652A (en) * | 2022-03-08 | 2022-05-17 | 北京工商大学 | Purification and crystal preparation method of Burkholderia ester synthetase Bur01 |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111675754A (en) * | 2020-05-19 | 2020-09-18 | 四川一埃科技有限公司 | Botulinum toxin protein crystal structure, crystal preparation method and resolution method |
| CN114507652A (en) * | 2022-03-08 | 2022-05-17 | 北京工商大学 | Purification and crystal preparation method of Burkholderia ester synthetase Bur01 |
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