CN109180818A - A method of inclusion body is purified using GEM - Google Patents
A method of inclusion body is purified using GEM Download PDFInfo
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- CN109180818A CN109180818A CN201810897135.0A CN201810897135A CN109180818A CN 109180818 A CN109180818 A CN 109180818A CN 201810897135 A CN201810897135 A CN 201810897135A CN 109180818 A CN109180818 A CN 109180818A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07K2319/00—Fusion polypeptide
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Abstract
The invention discloses a kind of methods using GEM purifying inclusion body, construct recombinant protein and AcmA fusion protein expression vector, inducing expression, which obtains recombinant protein, largely to be existed with inclusion bodies, inclusion body is dissolved using 8M urea, using GEM by the recombinant protein of dissolution the adsorbing separation from urea, then carry out renaturation.This method can use GEM simple and easy to get and isolate and purify the recombinant protein with AcmA from the inclusion body that urea dissolves, and have good protein salvage efficiency, while can simplify renaturing inclusion bodies process, simplify operation, reduce enzyme activity loss.
Description
Technical field
The present invention relates to a kind of methods using GEM purifying inclusion body.
Background technique
Escherichia coli are a kind of most common recombinant expression hosts, and, genetic background fast with the speed of growth is clear, exists
The advantages that a large amount of operational instrument.PET plasmid is transferred to Escherichia coli, it is different for inducing using IPTG and carrying out heterologous protein recombinant expression
The main stream approach of source expression aspect.But Escherichia coli as expressive host there is also some inevitable disadvantages, such as forgive
Modifying Capability etc. after body is formed, shortage is expressed, wherein the formation of inclusion body is a kind of most common, least evitable phenomenon.Packet
Contain the formation of body mainly due to Escherichia coli great expression heterologous protein, part albumen can not be folded correctly, be lured by reducing
Lead temperature, the concentration of reduction IPTG can reduce the formation of inclusion body, but not be avoided that the formation of inclusion body still.Inclusion body
With correct primary structure, but higher structure is incorrect, causes its insoluble and without bioactivity.Utilize denaturant
If inclusion body can sufficiently be dissolved denaturation by 8M urea, renaturation is then carried out again, can make the inclusion body without enzyme activity again
With all or part of bioactivity.Traditional inclusion body denaturation renaturation process takes a long time, while multistep being needed to grasp
Make.
Lactococcus lactis is a kind of bacterium of security level (GRAS).The C-terminal of the peptide glycan hydrolase A cmA of Lactococcus lactis
Albumen anchoring domain can be special be integrated on the cell wall of Lactococcus lactis, using this characteristic, means are boiled by acid
Most of albumen and DNA in Lactococcus lactis are removed, abiotic Gram-positive matrix (Gram-positive has been obtained
Enhancer matrix particles, GEM).Albumen is merged with the recombination of AcmA, specifically can be integrated to GEM
On.GEM anchoring AcmA fusion protein have the following characteristics that with good biological safety, have good mechanical performance and
With good albumen specific bond ability.GEM has and will combine isolated energy from urea with AcmA label recombinant protein
Power.
Summary of the invention
In order to solve the problems in the existing technology, the present invention provides a kind of method using GEM purifying inclusion body, gram
The problem of taking inclusion body denaturation renaturation process in the prior art to take a long time, while needing many more manipulations.
The technical scheme is that a kind of method using GEM purifying inclusion body, sequentially includes the following steps:
1) recombinant protein-AcmA fusion expression vector is constructed, e. coli bl21 is transferred to；
2) inducing expression is carried out using IPTG；
3) GEM is prepared；
4) after inducing expression product is broken, supernatant is combined with GEM mixing, is centrifugated；
5) 8M urea dissolves inclusion body；
6) GEM purifies and separates recombinant protein from the inclusion body that urea dissolves；
7) renaturation of inclusion body；
8) enzyme activity determination.
In step 1), using Lactococcus lactis NZ9000 genome as template, amplification obtain AcmA (GenBank:
ADJ59253.1) anchorage zone segment is connected on pET28a and obtains anchoring plasmid pET28a-AcmA, with bacillus amyloliquefaciens
BH072 genome is template, and amplification obtains the genetic fragment of alpha-amylase (GenBank:AJE77362.1), is connected to plasmid
On pET28a-AcmA, recombinant expression plasmid pET28a- α-amylase-AcmA is obtained, Escherichia coli are transferred to by chemical conversion
Expression vector BL21.
In step 2), the monoclonal for the isolated BL21 with plasmid that crosses is incubated overnight to obtain seed liquor, with
The inoculum concentration of 1%-5% is inoculated with, as bacterium solution OD600IPTG is added when reaching 0.6-0.9 to be induced, induced concentration 0.5mM-
2mM, inducing temperature are selected as 37 DEG C, and induction time is -24 hours 18 hours, that is mould for 50 μ g/mL cards of addition for all culture mediums
The LB culture medium of element.
In step 3), the M17 culture medium culture Lactococcus lactis NZ9000 to OD for containing 1% glucose is utilized600In 2.0-
Between 2.5, thalline were collected by centrifugation by 4000rpm, washs removal culture medium using PBS, thallus is resuspended in the HCl of 0.1M-0.2M
In, it boils 30-60 minutes, precipitating is collected by centrifugation in 8000rpm, is sufficiently washed using PBS, obtains GEM.
In step 4), the thallus after induction is collected by centrifugation in 4000rpm, will using the PBS washing removal culture medium of pre-cooling
Thallus is resuspended in the PBS of pre-cooling, sonicated cells, ultrasonication in 2 seconds, is paused within 4 seconds, and ultrasonic time is 20 minutes,
8000rmp is centrifuged 20 minutes and separates, and takes precipitating.
In step 5), obtained inclusion body precipitating is sufficiently dissolved using 8M urea, 12000rmp centrifugation takes supernatant i.e.
Forgive liquid solution for urea dissolution.
In step 6), liquid solution is forgiven into the urea dissolution that step 5 obtains and GEM is mixed, stirring combines 20- at room temperature
50 minutes, 12000rpm centrifuge separation was taken precipitating, is sufficiently cleaned using PBS to remove non-specific binding and urea.
In step 7), it will be combined in the GEM resuspension and PBS of recombinant protein, 4 DEG C of overnight renaturation.
In step 8), the enzyme activity of DNS method measurement recombinant protein is utilized.
Beneficial effects of the present invention: the present invention carries out recombinant protein and AcmA amalgamation and expression, and inducing expression is to forgive the bodily form
Formula exists, and using the GEM of preparation as a kind of material, the recombination egg of AcmA label will be had from the urea lysate of inclusion body
White purifies and separates, and renaturation is carried out, measure its enzyme activity.GEM can have AcmA from most of as a kind of new purified material
The recombinant protein of label purifies and separates from the urea liquid of inclusion body have biology living using available after renaturation solution renaturation
The recombinant protein of property.
Detailed description of the invention
Fig. 1 GEM purifies and separates urea dissolves inclusion body；Wherein M: albumen Marker；1: urea dissolves inclusion body；2,3,4,
5:GEM for the first time, it is secondary, three times, four adsorbing separation recombinant proteins；6: residual protein after absorption；
Enzyme activity after Fig. 2 renaturation.
Specific embodiment
The present invention is described in more detail in the following with reference to the drawings and specific embodiments.
Embodiment 1: the building and inducing expression of recombinant protein-AcmA fusion expression vector
1) design of primers
According to the sequence of the AcmA of Lactococcus lactis NZ9000, design primer AF and AR, primer add respectively HindIII and
XhoI restriction enzyme site,；According to the gene order of the alpha-amylase of bacillus amyloliquefaciens BH072, design primer α F and α R, primer
NcoI and HindIII restriction enzyme site is added respectively.
2) vector construction
Using the genome of Lactococcus lactis NZ9000 as template, the genetic fragment of AcmA, double digestion are obtained using PCR amplification
It is connect afterwards with plasmid pET28a, obtains plasmid pET28a-AcmA after sequencing is correct；With the gene of bacillus amyloliquefaciens BH072
Group is template, obtains the genetic fragment of alpha-amylase using PCR amplification, connect after double digestion with plasmid pET28a-AcmA, is sequenced
Plasmid pET28a- α-amylase-AcmA is obtained after correct.
Obtained recombinant plasmid is constructed to be transferred in e. coli bl21.
3) inducing expression
It is incubated overnight with the LB culture medium containing 50 μ g/mL kanamycins, seed liquor is prepared, according to the inoculum concentration of 1-5%
It is inoculated into the LB culture medium containing 50 μ g/mL kanamycins, shake culture 3-5 hours, as bacterium solution OD600Between 0.6-0.9
When, IPTG is added and is induced, induced concentration 0.5mM-2mM, inducing temperature is selected as 37 DEG C, and induction time is 18 hours-
24 hours.
4) ultrasonication
Thalline were collected by centrifugation by 4000rmp, washs precipitating using the PBS of pre-cooling, centrifugation is resuspended cell with the PBS of pre-cooling, surpasses
Sound is broken, ultrasonication in 2 seconds, pauses within 4 seconds, and ultrasonic time is 20 minutes, and 8000rmp is centrifuged 20 minutes and separates, and takes precipitating.
The present embodiment obtains the recombinant protein based on inclusion bodies.Inducing temperature and use IPTG concentration are to recombination egg
White distribution has a very big impact.Improve temperature, increase IPTG concentration can be improved expression rate, and then improve inclusion body
It is formed.Higher inducing temperature and higher IPTG concentration are used in the present embodiment, and most recombinant proteins can be made to wrap
Contain body form to exist.
Embodiment 2:GEM purifies and separates inclusion body
1) prepared by GEM
Utilize the M17 culture medium culture Lactococcus lactis NZ9000 to OD for containing 1% glucose600Between 2.0-2.5,
Thalline were collected by centrifugation by 4000rpm, washs removal culture medium using PBS, thallus is resuspended in the HCl of 0.1M-0.2M, is boiled
30-60 minutes, precipitating was collected by centrifugation in 8000rpm, is sufficiently washed using PBS, obtains GEM.
2) solubilization of inclusion bodies
Obtained inclusion body precipitating is sufficiently dissolved using 8M urea, 12000rmp centrifugation, taking supernatant is urea dissolution
Forgive liquid solution.
3) GEM purifies and separates recombinant protein
By supernatant obtained in the previous step, i.e., liquid solution is forgiven in urea dissolution and GEM is mixed, and stirring combines 20-50 at room temperature
Minute, 12000rpm centrifuge separation is taken precipitating, is sufficiently cleaned using PBS to remove non-specific binding and urea.For maximum
Degree recycles the recombinant protein being dissolved in urea, carries out 4 combinations altogether.
SDS-PAGE measures albumen distribution situation.
The recombinant protein of the present embodiment acquisition GEM purifies and separates.GEM has well with the binding ability of AcmA, is urinating
Still can be in conjunction with recombinant protein under this strong denaturant of element, and it is separated to pass through centrifugation.
Embodiment 3: recombinant protein renaturation
1) recombinant protein renaturation
The GEM for being combined with recombinant protein is resuspended in PBS, 4 DEG C of overnight renaturation.
12000rmp centrifuge separation, obtains active recombinant protein.
2) enzyme activity is identified
Utilize the enzyme activity of DNS method measurement recombinant protein.Measure 4 enzyme activity for combining the recombinant protein Jing Guo renaturation later.
The present embodiment obtains biologically active recombinant protein after renaturation, and measures enzyme activity.GEM will have the weight of AcmA
Histone is forgiven in liquid solution from urea dissolution to be isolated and purified out, is carried out renaturation as renaturation solution using PBS, is obtained having life
The active recombinant protein of object.
Although above in conjunction with attached drawing, invention has been described, and the invention is not limited to above-mentioned specific implementations
Mode, the above mentioned embodiment is only schematical, be not it is restrictive, those skilled in the art this
Under the enlightenment of invention, without breaking away from the scope protected by the purposes and claims of the present invention, many shapes can also be made
Formula, within these are all belonged to the scope of protection of the present invention.
Claims (9)
1. a kind of method using GEM purifying inclusion body, it is characterised in that described method and step is as follows:
1) recombinant protein-AcmA fusion expression vector is constructed, e. coli bl21 is transferred to；
2) inducing expression is carried out using IPTG；
3) GEM is prepared；
4) after inducing expression product is broken, supernatant is combined with GEM mixing, is centrifugated；
5) 8M urea dissolves inclusion body；
6) GEM purifies and separates recombinant protein from the inclusion body that urea dissolves；
7) renaturation of inclusion body；
8) enzyme activity determination.
2. a kind of method using GEM purifying inclusion body according to claim 1, it is characterised in that step 1) is with lactic acid cream
Coccus NZ9000 genome is template, and amplification obtains the anchorage zone AcmA segment, is connected on pET28a and obtains anchoring plasmid
PET28a-AcmA, using bacillus amyloliquefaciens BH072 genome as template, amplification obtains the genetic fragment of alpha-amylase, connects
Onto plasmid pET28a-AcmA, recombinant expression plasmid pET28a- α-amylase-AcmA is obtained, is transferred to greatly by chemical conversion
Enterobacteria expression vector BL21.
3. a kind of method using GEM purifying inclusion body according to claim 1, it is characterised in that step 2) refers to scribing line point
It from the monoclonal of the BL21 with plasmid is obtained, is incubated overnight to obtain seed liquor, is inoculated with the inoculum concentration of 1%-5%, works as bacterium solution
OD600IPTG is added when reaching 0.6-0.9 to be induced, induced concentration 0.5mM-2mM, inducing temperature is selected as 37 DEG C, induction
Time is -24 hours 18 hours, and all culture mediums are the LB culture medium for adding 50 μ g/mL kanamycins.
4. a kind of method using GEM purifying inclusion body according to claim 1, it is characterised in that step 3), which refers to utilize, to be contained
The M17 culture medium culture Lactococcus lactis NZ9000 to OD of 1% glucose600Between 2.0-2.5, bacterium is collected by centrifugation in 4000rpm
Body, using PBS wash removal culture medium, thallus is resuspended in the HCl of 0.1M-0.2M, is boiled 30-60 minutes, 8000rpm from
The heart collects precipitating, is sufficiently washed using PBS, obtains GEM.
5. a kind of method using GEM purifying inclusion body according to claim 1, it is characterised in that step 4) refers to
The thallus after induction is collected by centrifugation in 4000rpm, and using the PBS washing removal culture medium of pre-cooling, thallus is resuspended in the PBS of pre-cooling
In, sonicated cells, ultrasonication in 2 seconds pauses for 4 seconds, and ultrasonic time is 20 minutes, and 8000rmp is centrifuged 20 minutes and separates, and takes
Precipitating.
6. a kind of method using GEM purifying inclusion body according to claim 1, it is characterised in that step 5), which refers to, to be obtained
Inclusion body precipitating sufficiently dissolved using 8M urea, 12000rmp centrifugation, taking supernatant is that liquid solution is forgiven in urea dissolution.
7. a kind of method using GEM purifying inclusion body according to claim 1, it is characterised in that step 6) refers to step
Liquid solution is forgiven in 5 obtained urea dissolutions and GEM is mixed, and stirring combines 20-50 minutes at room temperature, 12000rpm centrifuge separation,
Precipitating is taken, is sufficiently cleaned using PBS to remove non-specific binding and urea.
8. a kind of method using GEM purifying inclusion body according to claim 1, it is characterised in that step 7), which will refer to, to be combined
Have in the GEM resuspension and PBS of recombinant protein, 4 DEG C of overnight renaturation.
9. a kind of method using GEM purifying inclusion body according to claim 1, it is characterised in that step 8) refers to utilization
The enzyme activity of DNS method measurement recombinant protein.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551703A (en) * | 2019-09-10 | 2019-12-10 | 天津大学 | Preparation and application methods of novel material BEM |
| CN110628746A (en) * | 2019-09-10 | 2019-12-31 | 天津大学 | Method for preparing BEM for purifying immobilized recombinant protein with AcmA |
| CN114317345A (en) * | 2021-12-28 | 2022-04-12 | 龙岩学院 | Preparation method of lactococcus lactis GEM particles |
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110551703A (en) * | 2019-09-10 | 2019-12-10 | 天津大学 | Preparation and application methods of novel material BEM |
| CN110628746A (en) * | 2019-09-10 | 2019-12-31 | 天津大学 | Method for preparing BEM for purifying immobilized recombinant protein with AcmA |
| CN114317345A (en) * | 2021-12-28 | 2022-04-12 | 龙岩学院 | Preparation method of lactococcus lactis GEM particles |
| CN114317345B (en) * | 2021-12-28 | 2023-12-01 | 龙岩学院 | Preparation method of lactococcus lactis GEM particles |
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