CN104711245A - Chondroitin sulfateyase ABC (ChSase ABC) mutant fusion protein and application thereof - Google Patents
Chondroitin sulfateyase ABC (ChSase ABC) mutant fusion protein and application thereof Download PDFInfo
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Abstract
The invention provides a mutational chondroitin sulfateyase ABC (ChSase ABC) mutant fusion protein and coding genes and preparation method thereof. The ChSase ABC mutant fusion protein provided by the invention is prepared by adopting a one-step purification method, the purification method is simple, and a ChSase ABC mutant with high purity and high activity is obtained. The obtained ChSase ABC has high enzyme activity and can be used for producing chondroitin sulfate, chondroitin sulfate producing conditions are not high, and a chondroitin sulfate producing method is simple and convenient and is easy to control.
Description
Technical field
The present invention relates to the chondroitinase ABC fusion rotein of rite-directed mutagenesis prepared by a kind of gene recombination method.Its key be chondroitinase ABC gene fixed point transformation, clone, express and the fermentation expression of product and separation and purification preparation.
Background technology
Chondroitinase (chondroitinase or chondroitin sulfateyase, referred to as " ChSase ") is the lyase that the glycosaminoglycan such as chondroitin sulfate, chrondroitin, hyaluronic acid can be degraded to unsaturated disaccharide (Δ Di and oligosaccharides) by a class.
Along with the further investigation to ChSase, have a wide range of applications value to find chondroitinase ABC (referred to as " ChSase ABC ").Scientific research personnel utilizes ChSase ABC detect chondroitin sulfate and produce low-molecular weight chondroitin sulfate.Bao Lunjun, Yang Jiancheng etc. detect the hyaluronic acid in shark's fin by the method for ChSase ABC enzymolysis-high performance liquid chromatography, adopt ZORBAX glycan analysis post, ultraviolet detection wavelength is 226nm, to detect in shark's fin hyaluronic massfraction be 0.86-1.96% (Bao Lunjun etc. the enzymolysis-high effective liquid chromatography for measuring of chondroitin sulfate in shark's fin. chromatogram, 2002,20 (6): 557-559).Zhu Yuning, Ao Lei etc. detect the content of CS equally by the method for enzymolysis high performance liquid chromatography, adopt Ultimate XB-NH2 post, ultraviolet detection wavelength is 232nm, moving phase is sodium-acetate buffer (pH5.6)-acetonitrile (950:50), flow velocity is 1.0mL/min, this chromatographic condition can by CS A, B, C separates (Zhu Yuning etc. the content of enzymolysis high effective liquid chromatography for measuring chondroitin sulfate. Chinese biochemical drug magazine, 2012,33 (6): 827-829).
ChSase ABC also has very large effect in clinical application in addition.In relieving retina sex change, degradable chondroitin sulfate proteoglycan (CSPGs), CSPGs is the important component of glial scar after central nervous system (CNS) damage, and neural axon can be suppressed to regenerate.CSPGs suppresses neurotization mainly through sugared ammonia polysaccharide chain, therefore removes GAG or disturbs its synthesis all can make axon regeneration.Topaz seedling, Gao Pengfen etc. are by intravitreal ChSase ABC enzyme, immunofluorescence technique observes the expression of CSPGs, reverse transcription-polymerase chain reaction (RT-PCR) method detects the expression of versican mRNA, detect sight sensor apoptosis situation, result shows the CSPGs of ChaseABC by degraded sex change rat retina abnormal deposition, suppress the apoptosis of photoreceptor cell, thus the reparation of promotion damage nethike embrane (topaz seedling etc. chondrosulphatase relieving retina sex change rat photoreceptor cell apoptosis. Yangzhou University's journal, 2012, 33 (4): 19-23.).
In raising hind limb motor ability, ChSase ABC can repair spinal injury.Spinal injury is the common complication of spinal fracture, quadriplegia can be caused, a series of severe complications such as gatism and respiratory muscle paralysis, neural regeneration after spinal cord injury and function reparation, be a great problem in current medical field, ChSase ABC can degrade CSPGs.Sun Yongxin, Liu Ning etc. utilize ChSase ABC to combine the impact of hyperbaric oxygen preconditioning on acetylcholinesterase (AChE) content in different times hind limb motor function after spinal cord of adult rats damage and gastrocnemius muscle motor end-plate, detect the effect of ChSase ABC.Result shows that hyperbaric oxygen preconditioning can be improved the motor function of rat suffering limb and improve the activity of AChE, associating ChSase ABC effect stronger (Sun Yong Xinhua etc. chondroitinase ABC associating hyperbaric oxygen preconditioning is on the impact of rats with spinal cord injury hind limb motor function. dissect scientific advance, 2012,18 (6): 526-529.).Nicole J.Tester etc. treats injured cat with ChSaseABC, ChSase ABC has repaired the spinal injury of cat, improve the motor capacity of cat (see Nicole J.et al.Chondroitinase ABC improves basic andskilled locomotion in spinal cord injured cats.Experimental Neurology, 2008,209 (2): 483-496).
In recent years, Mark R Brown etc. finds after deliberation, main ingredient in Lumbar intervertebral disc protrusion is proteoglycan, then can fall outstanding intraspinal glycosaminoglycan, reduce intraspinal pressure by selective degradation with ChSase ABC and ChSase AC, simultaneously to the harmless (Brown of canalis spinalis peripheral tissues, M D.Method for treating intervertebral disc displacement with enzymes [P]: US, 4696816,1987-09-29).Calendar year 2001 Denholm EM etc. find the formation of ChSase AC, ChSase B energy check melanin knurl blood vessel and (the Denholm E M such as the transfer of oncocyte and hyperplasia, Lin Y Q, Silver P J.Anti-tumor activities of chondroitinaseAC and chondroitinase B:inhibition of angiogenesis, proliferation andinvasion.European Journal of Pharmacology, 2001,416 (3): 213-221).LeeMC etc. find after cartilage is with ChSase ABC process, and the chondrocyte of transplanting and the Adhering capacity of the cartilage surface of a wound strengthen greatly; (Lee M C, Sung K L, Kurtis M S, et a1.Adhesiveforce of chondrocytes to cartilage effects of chondroitinase ABC.ClinOrthop, 2000,370 (1): 286-294).
In addition chondroitinase ABC also can be used for enzymolysis production chondroitin sulfate.Acid-hydrolysis method, ion exchange method and enzymolysis process are often adopted to the preparation of low-molecular weight chondroitin sulfate.First two method needs more complicated experimental installation and polluting the environment, and comparatively speaking, the condition that enzymolysis process needs is not high, method is easy and easily control.The key of enzymolysis process is to obtain a large amount of chondroitinases.The method of separation and purification chondroitinase is very complicated, usually needs the chromatogram purification through multistep, and yield is very low.Heterologous recombination is expressed ChSase ABC and is commonly used to one of means improving ChSase ABC output.But very limited to the heterologous recombination expression study of ChSase ABC, the up to the present domestic report also not having this respect.Only have Pojasek etc. in E.coil, have expressed the gene (chsase gene) of chondrosulphatase AC enzyme and B enzyme, and separation and purification is to activated protein, in its research, carrier used is pET15b and pCRT7/NT, and the fusion tag in these two carriers is His-tag.But there is obvious shortcoming in His-tag, namely the avidity with affiliation carrier is not strong, the solubility of conjugated protein with it can not be increased, be difficult to improve purification effect and protein solubility ratio etc. (see Kevin Pojasek, Zachary Shriver, Patrick Kiley, Ganesh Venkataraman and Ram Sasisekharan.Recombinant Expression, Purification and Kinetic Characterization of Chondroitinase AC andChondroitinase B from Flavobacterium hparinum.Biochemical andBiophysical Research Communications, 2001, 286:343-351).
Patent application CN103305496A discloses a kind of method extracting chondrosulphatase from microorganism, but the method extraction step is loaded down with trivial details, and cost is higher.
Summary of the invention
For solving the deficiencies in the prior art, the present invention adopts the method for amalgamation and expression and rite-directed mutagenesis, obtains the fusion rotein of the ChSase ABC mutant of great amount of soluble, further increasing ChSase ABC enzyme and lives; And adopting the method for single step purification, purification process is simple, and obtains highly purified ChSase ABC mutant.
Main purpose of the present invention is to provide a kind of chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein, wherein, described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein comprises chondroitin sulfate A (CSA) BC enzyme mutant and maltose binding protein, wherein, described chondroitin sulfate A (CSA) BC enzyme mutant has the aminoacid sequence shown in SEQ ID NO:1, and described maltose binding protein is held with described chondroitin sulfate A (CSA) BC enzyme N by peptide section connection portion and is connected.The invention provides a kind of chondroitin sulfate A (CSA) BC enzyme fusion proteins of rite-directed mutagenesis, it is characterized in that the amino acid mutation of Asn771 position being Ala.
Preferably, in chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein of the present invention, described maltose binding protein has the aminoacid sequence shown in SEQ ID NO:2.
Preferably, in chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein of the present invention, described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein has the aminoacid sequence shown in SEQ ID NO:3.
Preferably, in chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein of the present invention, take chondroitin sulfate A (CSA) as substrate, the enzyme of described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein is lived and is lived than enzyme and is respectively 24.96IU/mL enzyme and 31.32IU/mg protein, take chondroitin sulfate B as substrate, described chondroitin sulfate A (CSA) BC enzyme mutant fusion protease is lived and is lived than enzyme and is respectively 21.58IU/mL enzyme and 27.07IU/mg protein.
Preferably, in chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein of the present invention, the V of described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein
maxand k
cat/ K
m, when taking chondroitin sulfate A (CSA) as substrate, be respectively 22.52 μm of ol/Ls and 50.25L/ μm of ols; When taking chondroitin sulfate B as substrate, it is respectively 4.59 μm of ol/Ls and 12.22L/ μm of ols.
Another object of the present invention is to provide a kind of DNA, the chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein described in described DNA sequence encoding.
Another object of the present invention is to provide a kind of recombinant vectors, and described recombinant vectors comprises carrier and described coding chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein DNA, and wherein said carrier comprises pMAL-c2x.
Another object of the present invention is to provide a kind of transformant, and described transformant is the transformant described recombinant vectors being imported host cell and obtains, and wherein, described host cell comprises E.coliBL21 (DE3).
An also object of the present invention is to provide a kind of method preparing described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein, and described preparation method comprises: (1) use PCR by described in obtaining with the primer in mutational site the recombinant vectors of pMAL-c2x-ChSase ABC I mutant; (2) transform described recombinant vectors in described step (1) to described host cell, express described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein; And (3) broken described transformant, use the chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein of expressing described in maltose binding protein affinity protein purification.
Of the present invention also measured were sudden change after the optimum temperuture of chondroitin sulfate A (CSA) BC enzyme fusion proteins be 45 degree and optimal pH be 7.5.
The present invention determines the kinetic constant of the chondroitin sulfate A (CSA) BC enzyme fusion proteins after sudden change equally, comprising V
maxand k
cat/ K
m, when taking chondroitin sulfate A (CSA) as substrate, it is respectively 22.52 μm of ol/Ls and 50.25L/ μm of ols, and when taking chondroitin sulfate B as substrate, it is respectively 4.59 μm of ol/Ls and 12.22L/ μm of ols.
The present invention also determines the thermostability of the chondroitin sulfate A (CSA) BC enzyme fusion proteins after sudden change equally, comprising at 45 DEG C, 40 DEG C, 35 DEG C and 30 DEG C, at 30 DEG C and 35 DEG C, the enzyme that can retain 90% after 210min is lived, at 40 DEG C, can retain the enzyme work of 72% after 210min, at 45 DEG C, 210min can retain the enzyme work of 41%.
Asn771 amino acid mutation is become Ala by the method for rite-directed mutagenesis by the present invention, take chondroitin sulfate A (CSA) as substrate, the enzyme of Asn771Ala is lived and is lived than enzyme and is respectively 24.96IU/mL enzyme and 31.32IU/mg protein, take chondroitin sulfate B as substrate, enzyme is lived and is lived than enzyme and is respectively 21.58IU/mL enzyme and 27.07IU/mg protein.The MBP-ChSase ABC I do not suddenlyd change, take chondroitin sulfate A (CSA) as substrate, enzyme is lived and is lived than enzyme and is respectively 20.18IU/mL enzyme and 22.52IU/mg protein, take chondroitin sulfate B as substrate, and enzyme is lived and lived than enzyme and is respectively 13.08IU/mLenzyme and 14.60IU/mg protein.
Preferably, the present invention prepares in the method for described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein, and in described step (3), maltose binding protein affinity chromatography comprises use amylose resin, the separation of yam starch post.
Present invention also offers a kind of method of producing low-molecular weight chondroitin sulfate, described production method comprises: the chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein described in use produces low-molecular weight chondroitin sulfate.
Accompanying drawing explanation
Fig. 1 is the pMAL-c2x-ChSase ABC I do not suddenlyd change is template, the pMAL-c2x-ChSase ABC I electrophoretogram of the sudden change using the primer containing mutational site to obtain through pcr amplification, swimming lane M is: Marker, and swimming lane 1 is 60 DEG C of PCR primer being annealing temperature.
Fig. 2 is the protein electrophoresis figure expressed, wherein, swimming lane M is marker (from top to bottom molecular weight is 250kDa, 150kDa, 100kDa, 70kDa successively), the soluble protein that swimming lane 1 is blank, swimming lane 2 is E.coli BL21 (DE3)/pMAL-ChSase ABC, arrow indication place is fusion rotein MBP-ChSase ABC I mutant (140kDa).
in sequence table:
Sequence 1 is the aminoacid sequence of chondroitin sulfate A (CSA) BC enzyme mutant;
Sequence 2 is intestinal bacteria (Escherichia coli) maltose binding protein aminoacid sequence;
Sequence 3 is chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein aminoacid sequence of the present invention;
Sequence 4 is chondroitinase ABC enzyme mutant fusion rotein DNA sequence dna of the present invention;
Sequence 5 is introduce the upstream primer nucleotide sequence for pcr amplification pMAL-c2x-ChSase ABC I mutant in mutational site;
Sequence 6 is introduce the downstream primer nucleotide sequence for pcr amplification pMAL-c2x-ChSase ABC I mutant in mutational site.
Embodiment
Further describe the present invention below in conjunction with specific embodiment, advantage and disadvantage of the present invention will be more clear along with description.But these embodiments are only exemplary, do not form any restriction to scope of the present invention.It will be understood by those skilled in the art that and can modify to the details of technical solution of the present invention and form or replace down without departing from the spirit and scope of the present invention, but these amendments and replacement all fall within the scope of protection of the present invention.
Below, embodiment is herein specifically described.
chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein
The chondroitin sulfate A (CSA) BC enzyme fusion proteins of the sudden change related to herein is (following, sometimes also referred to as " MBP-ChSase ABC "), it merges the chondroitin sulfate A (CSA) BC enzyme and maltose binding protein (following, sometimes also referred to as " MBP ") that there are sudden change.
The chondroitin sulfate A (CSA) BC enzyme related to herein can be any chondrosulphatase, is preferably selected from the chondrosulphatase that proteus vulgaris (Proteus vulgaris), Aeromonas hydrophila (Aeromonas liquefaciens) and Flavobacterium heparinum (Flavobacterium heparinum) are originated.According to a preferred embodiment herein, chondroitin sulfate A (CSA) BC enzyme is the chondrosulphatase in proteus vulgaris source; Especially, chondrosulphatase is the chondroitin sulfate A (CSA) BC enzyme of the proteus vulgaris eliminating coded signal peptide base, and is obtained the chondroitin sulfate A (CSA) BC enzyme of sudden change by rite-directed mutagenesis.According to this paper preferred embodiment, chondroitin sulfate A (CSA) BC enzyme mutant has the amino acid shown in SEQ ID NO:1 herein.
In chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein, chondroitin sulfate A (CSA) BC enzyme mutant directly and maltose binding protein fusion.
The maltose binding protein related to herein can be the maltose binding protein that any those of ordinary skill in the art can obtain.Preferably come from colibacillary maltose binding protein.From colibacillary natural MBP can with maltose Specific adsorption, participate in intestinal bacteria to the transhipment of maltose and utilization.MBP not only can be combined with amylose starch, realizes Human serum protein, also can realize Human serum protein with yam starch, thus greatly can reduce the separation and purification cost of enzyme, is conducive to the separation and purification realizing industrially scalable.In a preferred embodiment, maltose binding protein herein has the amino acid shown in SEQ ID NO:2.
Preferably, the chondroitin sulfate A (CSA) BC enzyme fusion proteins of the sudden change of this paper has the amino acid shown in SEQ ID NO:3.
DNA
Also relate to a kind of DNA of chondroitin sulfate A (CSA) BC enzyme fusion proteins of above-mentioned all sudden changes of encoding herein, it is the encoding sequence of this fusion rotein.
For the nucleotide sequence being meant to the aminoacid sequence of directly specifying its protein product of the term " encoding sequence " of this specification sheets.
The border of encoding sequence determines by opening frame usually, described in open frame and usually start with ATG initiator codon or alternative initiator codon such as GTG and TTG, and to terminate with terminator codon such as TAA, TAG and TGA.Encoding sequence can be DNA, cDNA or recombinant nucleotide sequence.
According to this paper preferred embodiment, DNA has the base sequence shown in SEQ ID NO:4.
recombinant vectors
Also relate to mutational vector herein, it comprises the above-mentioned chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein of coding.
The recombinant vectors related to herein comprise above-mentioned all fusion roteins of encoding DNA sequence dna, promotor, transcribe and translation termination signal.DNA sequence dna described herein can combine with other regulating and controlling sequence, produces recombinant vectors, this carrier can comprise one or more (several) easily restriction site to allow to insert in these sites or replace the DNA sequence dna of encoded peptide section.Alternative, DNA sequence dna herein can be expressed by inserting the DNA sequence dna comprising described aminoacid sequence in the suitable carrier for expressing.In the process preparing recombinant vectors, encoding sequence is imported in carrier, thus this encoding sequence is operably connected with suitable expression regulation sequence.
Promotor, transcription signal, translation termination signal and other regulating and controlling sequence are that any those of ordinary skill in the art can select according to routine and determine.
Recombinant vectors can be any carrier (such as, plasmid or virus), and it can carry out recombinant DNA step easily, and can produce the expression of nucleotide sequence.The selection of carrier will usually depend on carrier and will introduce the consistency of the host cell of this carrier.Carrier can be wire or closed hoop plasmid.
Carrier can be autonomously replicationg vector, such as, and plasmid, extra-chromosomal element, minichromosome or artificial chromosome.Carrier can contain any structure for guaranteeing self replication.Or carrier can be a kind of when being introduced in host cell, to be incorporated in genome and the carrier copied together with the karyomit(e) incorporating this carrier.In addition, can use independent carrier or plasmid or two or more carriers or plasmid, its global DNA jointly containing host cell gene group to be introduced, maybe can use transposon.
According to this paper preferred embodiment, recombinant vectors is herein pMAL-c2x.Preferably, this mutational vector is built by following steps:
With the pMAL-c2x-ChSase ABC I do not suddenlyd change for template, with SEQ ID NO:5 and SEQ ID NO:6 for primer, PCR obtains pMAL-c2x-ChSase ABC I mutant.
transformant
Also relate to transformant herein, it is the transformant obtained comprising the above-mentioned chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein DNA importing host cell of coding.
The transformant related to herein can be used for the production of protein, by the vector introduction host cell comprising DNA sequence dna is herein obtained this transformant, and in suitable substratum, cultivates this transformant, the target protein that can be used for required for production.
Term " host cell " comprises the spawn of mother cell, and it is different from mother cell due to the sudden change occurred in reproduction process.Host cell can be any cell useful in the restructuring of peptide section herein produces, such as, and protokaryon or eukaryotic cell.Prokaryotic host cell can be any gram positive bacterium or gram negative bacterium.Gram positive bacterium includes but not limited to, bacillus, streptococcus, streptomyces, Staphylococcus, enterococcus spp, genus lactubacillus, lactococcus, fusobacterium, ground bacillus belong to and bacillus marinus genus.Gram negative bacterium includes but not limited to, intestinal bacteria, Rhodopseudomonas, salmonella, campylobacter, Helicobacterium, Flavobacterium, Fusobacterium, mud Bacillaceae, eisseria and Ureaplasma.Bacterial host cell can be any bacillus cell.In enforcement herein, useful bacillus cell includes but not limited to, Alkaliphilic bacillus, bacillus amyloliquefaciens, bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, bacillus firmus, bacillus lautus, bacillus lentus, Bacillus licheniformis, bacillus megaterium, bacillus pumilus, bacstearothermophilus, subtilis and Bacillus thuringiensis cell.Bacterial host cell can also be any streptococcus cell.In enforcement herein, useful streptococcus cell includes but not limited to, streptococcus equisimilis, streptococcus pyogenes, streptococcus uberis and zooepidemicus cell.Bacterial host cell can also be any Streptomyces cell.In enforcement herein, useful Streptomyces cell includes but not limited to, not streptomyces chromogenes, deinsectization streptomycete, streptomyces coelicolor, streptomyces griseus and shallow Streptomyces glaucoviolaceus cell.Host cell can also be eukaryote, as Mammals, insect, plant or fungal cell.In concrete at one, host cell is fungal cell.In concrete at one, fungal host cells is yeast cell.In another is concrete, fungal host cells is filamentous fungal cells.
According to this paper preferred embodiment, bacterial host cell is e. coli bl21 (DE3).
the method of the chondroitin sulfate A (CSA) BC enzyme fusion proteins of sudden change
Also relate to a kind of method preparing chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein herein, comprise
(i). the pMAL-c2x-ChSase ABC I do not suddenlyd change plasmid vector is provided;
(ii). with SEQ ID NO:5 and SEQ ID NO:6 for primer, PCR obtains the pMAL-c2x-ChSase ABC I mutational vector after sudden change.
The N end of the chondroitin sulfate A (CSA) BC enzyme mutant related to herein is combined with maltose binding protein by peptide section connection portion.
the method of purified chondroitin sulfates ABC enzyme mutant fusion rotein
Also relate to the method for purified chondroitin sulfates ABC enzyme mutant fusion rotein herein, it comprises:
A. amylose resin is utilized to adsorb the step of the crude product of the chondroitin sulfate A (CSA) BC enzyme fusion proteins containing sudden change, and
B. the step of maltose eluting chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein is utilized.
The method of the purified chondroitin sulfates ABC enzyme mutant fusion rotein related to herein, before described a and b, the method also comprises:
The pMAL-c2x-ChSase ABC I do not suddenlyd change plasmid vector is provided;
PCR obtains the pMAL-c2x-ChSase ABC I mutational vector of sudden change;
Recombinant vectors is imported host cell and obtains transformant; And
Utilize this transformant of culture medium culturing and obtain the crude product of described sulfur acid Chondroitin A BC enzyme mutant fusion rotein.
Wherein, the cultivation of transformant can use the conventional nutrient culture comprising carbon source, nitrogenous source, inorganic salt, various VITAMIN etc. to carry out, as carbon source, the carbohydrates such as such as glucose, sucrose, fructose, maltose can be used, the alcohols such as ethanol, methyl alcohol, the organic acids such as citric acid, oxysuccinic acid, succsinic acid, toxilic acid, fumaric acid, waste molasses etc.As nitrogenous source, such as ammonia, ammonium sulfate, ammonium chloride, ammonium nitrate, urea etc. can be used alone or as a mixture.In addition, as inorganic salt, such as potassium phosphate,monobasic, potassium primary phosphate, magnesium sulfate etc. can be used.In addition, the nutrient substances such as various VITAMIN such as peptone, meat extract, yeast extract, corn steep liquor, casamino acids, vitamin H can be added in substratum.
Cultivate and usually carry out under the aerobic condition such as aeration-agitation, vibration.Culture temperature is not particularly limited, as long as host microorganism can the temperature of growth reproduction, in addition, the pH in culturing process is also not particularly limited, as long as host microorganism can the pH of growth reproduction.PH adjustment in cultivation can by adding acid or alkali carries out.
According to this paper preferred embodiment, cultivating above-mentioned transformant is the cultivation carried out under the condition of induction, expresses thus and obtaining chondroitin sulfate A (CSA) BC enzyme mutant.The condition of inducing culture is: the IPTG of 0.5mM.In a further preferred embodiment, above-mentioned inducing culturing condition is: 10-42 DEG C of inducing culture 15-28 hour.In preferred at one, above-mentioned inducing culturing condition is: 16 DEG C of inducing culture 20 hours.
According to this paper preferred embodiment, the solvent carrying out the substratum of above-mentioned inducing culture is water, and solute is 10g/L NaCl, 5g/L yeast extract, 10g/L peptone and 100 μ g/L penbritins.
For the method for crude product preparing sulfur acid Chondroitin A BC enzyme mutant fusion rotein, be not particularly limited, various methods can be used.Usually, can prepare as follows: the transformant utilizing above-mentioned suitable culture medium culturing, by using the physical methods such as ultrasonic wave, squeezing, osmotic shock, or make use of the chemical processes such as tensio-active agent, or ferment treatment etc. are broken or solubilized, and the operation such as centrifugation or filtration, thus obtain the crude product of sulfur acid Chondroitin A BC enzyme mutant fusion rotein.
According to this paper preferred embodiment, the physical methods such as ultrasonic wave, squeezing, osmotic shock are adopted by the transformant culture thalline of above-mentioned cultivation to carry out fragmentation and obtain the crude product of sulfur acid Chondroitin A BC enzyme mutant fusion rotein.
According to this paper preferred embodiment, ultrasonication is adopted to obtain the crude product of the chondroitin sulfate A (CSA) BC enzyme fusion proteins containing sudden change.
According to this paper preferred embodiment, the crude product being above-mentioned sulfur acid Chondroitin A BC enzyme mutant fusion rotein is realized a step Human serum protein by amylose resin (amylose resin).Amylose resin is that any those of ordinary skill in the art can select according to routine and determine, can use the amylose resin that can be purchased, such as MBP Trap HP (article number 28-9187-78, GE Healthcare.In a preferred embodiment, be 1mL/min by the flow velocity of the supernatant liquor of amylose resin.In a preferred embodiment, the concentration for the maltose of wash-out is 10mM.In a preferred embodiment, the flow velocity for the maltose of wash-out is 1mL/min.In a preferred embodiment, this amylose resin is through pre-equilibration.
Embodiment
Below in conjunction with specific embodiment to being described further, but be not limited to following examples herein herein.
In following embodiment, if no special instructions, ordinary method is.Described primer synthesis and examining order complete by Hua Da gene, Q5
tMhigh-Fidelity 2 × Master Mix and all Dpn I is purchased from New England Biolabs company; All competent cells (as: DH5 α and BL21 (DE3)) are purchased from Beijing Bo Maide Bioisystech Co., Ltd); Chondroitin sulfate A (CSA) BC enzyme enzyme activity determination substrate chondroitin sulfate A (CSA) and B are purchased from Nanjing Olympic Duo Fu Buddhist nun bio tech ltd, and other pharmaceutical chemicals is general analysis pure reagent, purchased from Beijing Chemical Plant.
the table of embodiment 1, chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein (MBP-ChSase ABC)
reach
One, the acquisition of the pMAL-c2x-ChSase ABC I mutational vector of sudden change
The detailed process that mutational vector pMAL-c2x-ChSase ABC I obtains is as follows:
1, the design of primer and synthesis
DNA sequence dna (the Tam K.W.Study of chondroitin sulphate abc lyases and their use incombination for promotion of neurite growth.University of Hong Kong of proteus vulgaris chondroitin sulfate A (CSA) BC enzyme is obtained through Genbank inquiry, 2010.) the upstream and downstream primer that, sudden change Asn771Ala is used is respectively:
Upstream primer P1:5 '-GCCATTACTCCAACATTA
gCtACCCTTTGG-3 ' (SEQ ID NO:5) (what be with underscore is mutating alkali yl), downstream primer P2:5 '-
gCtAATGTTGGAGTAATGGCATGTTGGAATAAG-3 ' (SEQ ID NO:6) (what be with underscore is mutating alkali yl).
2, PCR obtains pMAL-c2x-ChSase ABC I mutant
The reaction system of pcr amplification is: 50ng proteus vulgaris genomic dna masterplate, and 100pmol often plants primer, 2 × Master Mix of response volume; Amplification program is: 98 degrees Celsius of denaturations 30 seconds, 98 degrees Celsius of sex change 7 seconds, and 60 degrees Celsius of primer annealings 30 seconds, 72 degrees Celsius extend 5 minutes, and after 30 circulations, 72 degrees Celsius extend 10 minutes and terminate reaction.This PCR result as shown in Figure 1, show to increase and obtain the pMAL-c2x-ChSase ABC I of 9.6kb, PCR uses Dpn I after obtaining plasmid, 37 degrees Celsius of digestions are the pMAL-c2x-ChSase ABC I template of not suddenling change, be transformed in DH5 α after digestion, and check order, sequencing result shows, successfully Asn771 is mutated into Ala.In Fig. 1, swimming lane M is molecular weight marker (stripe size is followed successively by 10kb, 8kb, 6kb, 5kb, 4kb, 3kb, 2kb, 1kb), swimming lane 1 is the pMAL-c2x-ChSase ABC I mutant obtained that increases, arrow indication place is 9.6kb target fragment, shows that successful PCR obtains pMAL-c2x-ChSase ABC I mutant.
Two, the expression of the fusion rotein MBP-ChSase ABC of chondroitin sulfate A (CSA) BC enzyme mutant
Plasmid in the bacillus coli DH 5 alpha of the pMAL-c2x-ChSase ABC containing sudden change in extraction step one, conventionally transforms colibacillus BL21 (DE3).Bacterium colony PCR qualification is carried out through penbritin screening and the primer that utilizes step 1 in step one to provide, obtain the e. coli bl21 (DE3) of the pMAL-c2x-ChSase ABC containing sudden change, namely BL21 (DE3)/pMAL-ChSase ABC is as the engineering bacteria of expressing the MBP-ChSase ABC I suddenlyd change.
With plasmid pMAL-c2x transformation of E. coli BL21 (DE3), obtain empty vector control BL21 (DE3)/pMAL-c2x.
By empty vector control and engineering bacteria respectively at the LB substratum (NaCl10g/L containing amicillin resistance, yeast extract is 5g/L, peptone 10g/L, containing 100 μ g/L penbritins) 37 degrees Celsius cultivate after 2.5 hours, adding final concentration is 0.5mM IPTG 16 degrees Celsius induction 20 hours.6000rpm, 6 minutes collected by centrifugation thalline also wash twice with 20mM Tris-HCl (pH 7.4), then resuspended thalline.(output rating is 300W re-suspension liquid to be carried out ultrasonication, ultrasonic 5 seconds and intermittently 6 seconds at every turn, process total time was 15 minutes), 6000rpm, 6 minutes centrifugal, and after ultrasonication, the supernatant liquor of centrifugal gained is the crude product of sulfur acid Chondroitin A BC enzyme mutant.
embodiment 2, by amylose starch column purification chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein
The present embodiment utilizes fusion partner (fusion partner) maltose binding protein MBP can realize a step with the affine absorption of amylose starch and is separated.Concrete Human serum protein step is as follows: be centrifugal 6 minutes of the 0.5mM IPTG abduction delivering thalline 100mL of 20 hours, 6000rpm by final concentration; Establish the phage control of non-abduction delivering simultaneously.Then operate respectively by following two schemes:
Wash twice with column equilibration liquid Column buffer (20mM Tris-HCl, 200mM NaCl, pH7.4), be resuspended in 30mL Column buffer, carry out ultrasonication (output rating is 300W, and ultrasonic 5 seconds and intermittently 6 seconds at every turn, process total time was 15 minutes).
Centrifuged supernatant by the amylose starch Human serum protein post of 1mL pre-equilibration, is collected by 10mM 1mL/min maltose wash-out with 1mL/min.
Target protein, can by target protein wash-out under 2 column volumes with 10mM maltose after amylose starch (amylose) resin absorption.Result as shown in Figure 2, shows that target protein can account for more than 90% after amylose resin single step purification.In Fig. 2, swimming lane M is marker (from top to bottom molecular weight is 250kDa, 150kDa, 100kDa, 70kDa successively), swimming lane 1 is blank, swimming lane 2 is E.coli BL21 (DE3)/soluble protein of pMAL-ChSase ABC mutant, and arrow indication place is MBP-ChSase ABC I mutant fusion protein (140kDa).
The detection of enzyme activity (unit is IU/mL) adopts the optical absorption method of 232nm, and the enzyme of 1IU is lived and is defined as the reaction effect that 37 centigrade per minutes produce 1 μm of oL unsaturated disaccharide.Chondroitin sulfate A (CSA) or B are mixed with the substrate solution of 1mg/mL, get substrate solution 2mL, add the purified product of 50 μ L of amylose starch column purification gained, final reaction volume is 2.05mL, surveys the absorbancy change △ A of unit time inherent 232nm
232.Extinction coefficient epsilon=3800M
-1.The ratio of enzyme activity and protein concentration (unit is mg/L) is defined as than enzyme (unit is IU/mg albumen) alive.Protein concentration monitoring adopts conventional Bradford method.
To sum up, by rite-directed mutagenesis, the enzyme of MBP-ChSase ABC I is lived to improve: take chondroitin sulfate A (CSA) as substrate, the enzyme of Asn771Ala is lived and is lived than enzyme and is respectively 24.96IU/mL enzyme and 31.32IU/mg protein, take chondroitin sulfate B as substrate, enzyme is lived and is lived than enzyme and is respectively 21.58IU/mL enzyme and 27.07IU/mg.The MBP-ChSase ABC I do not suddenlyd change, take chondroitin sulfate A (CSA) as substrate, enzyme is lived and is lived than enzyme and is respectively 20.18IU/mL enzyme and 22.52IU/mg protein, take chondroitin sulfate B as substrate, and enzyme is lived and lived than enzyme and is respectively 13.08IU/mL enzyme and 14.60IU/mg protein.
The present invention also achieves the single step purification of this fusion rotein by Human serum protein.With the protamine precipitation used in prior art (with reference to ANA LYDIA TKALEC, the people such as DOMINIQUEFINK, Isolation and Expression in Escherichia coli of cslA and cslB, Genes Coding for the Chondroitin Sulfate-Degrading EnzymesChondroitinase AC and Chondroitinase B, Respectively, fromFlavobacterium heparinum.APPL.ENVIRON.MICROBIOL.2000, 66 (1): 29-35) compare (see table 1) with a step column purification method, greatly can shorten the time that purifying needs, and purifying equipment used obviously reduces, reduce the production cost of purified chondroitin sulfates enzyme.Utilize the purifying of amylose resin only to need to carry out 2 hours, can obtain and can be used in industrial chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein.Purity reaches adaptable more than 95%.
Table 1 protamine precipitation is compared with the present invention one step column purification method
| Ordinary method | Single step purification method | |
| Purification step | Four steps (protamine precipitation+three step column purification) | One step |
| Purification time | About 2 days | 2 hours |
| Purity | >99％ | >95％ |
| Equipment | Complicated | Simply |
After bacterial strain is optimized, e. coli bl21 (DE3) (pMAL-c2x-ChSase ABC I) mutant strain, after cultivating 2.5 hours at 37 degrees Celsius, add 0.5mM IPTG, inducing temperature 16 degrees Celsius, the chondroitin sulfate A (CSA) BC enzyme mutant enzyme work of producing can reach 7488IU/L fermented liquid (taking chondroitin sulfate A (CSA) as substrate), and expression amount can reach 620mg/L fermented liquid, can reach 31.32IU/mg (taking chondroitin sulfate A (CSA) as substrate) than enzyme work.
The present invention plays a significant role in the production of chondroitin sulfate A (CSA) BC enzyme, and can be applied to each field in the same manner as chondroitin sulfate A (CSA) BC enzyme by the chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein that the present invention produces, and enzymic activity is higher.
Below describe the preferred embodiments of the present invention comprehensively, but various substituting and amendment can be carried out to them.Therefore, scope of the present invention should do not decided with reference to above description, but scope of the present invention should be decided with reference to appended claims and whole equivalent thereof.Any feature, whether whether (no matter being preferred) all with any other feature (no matter being preferred) can combine.Claims of the present invention should not be understood to the restriction with method+function, unless listed this type of restriction clearly by term " ... method " in a certain claim.
Claims (10)
1. a chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein, wherein, described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein comprises chondroitin sulfate A (CSA) BC enzyme mutant and maltose binding protein, wherein, described chondroitin sulfate A (CSA) BC enzyme mutant has the aminoacid sequence shown in SEQ ID NO:1, and described maltose binding protein is held with described chondroitin sulfate A (CSA) BC enzyme N by peptide section connection portion and is connected.
2. chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein according to claim 1, wherein, described maltose binding protein has the aminoacid sequence shown in SEQ ID NO:2.
3. chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein according to claim 1, wherein, described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein has the aminoacid sequence shown in SEQ ID NO:3.
4. chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein according to claim 1, wherein, take chondroitin sulfate A (CSA) as substrate, the enzyme of described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein is lived and is lived than enzyme and is respectively 24.96IU/mL enzyme and 31.32IU/mg protein, take chondroitin sulfate B as substrate, described chondroitin sulfate A (CSA) BC enzyme mutant fusion protease is lived and is lived than enzyme and is respectively 21.58IU/mL enzyme and 27.07IU/mg protein.
5. a DNA, the chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein described in described any one of DNA sequence encoding Claims 1 to 4.
6. a recombinant vectors, described recombinant vectors comprises carrier and coding chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein DNA according to claim 5, and wherein said carrier comprises pMAL-c2x.
7. a transformant, described transformant is transformant recombinant vectors according to claim 6 being imported host cell and obtains, and wherein, described host cell comprises E.coli BL21 (DE3).
8. prepare a method for chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein described in any one of Claims 1 to 4, described preparation method comprises:
(1) use PCR by described in obtaining with the primer in mutational site the recombinant vectors of pMAL-c2x-ChSase ABC I mutant;
(2) transform described recombinant vectors in described step (1) to described host cell, express described chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein; And
(3) broken described transformant, uses the chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein of expressing described in maltose binding protein affinity protein purification.
9. preparation method according to claim 8, wherein, in described step (3), maltose binding protein affinity chromatography comprises use amylose resin, the separation of yam starch post.
10. produce a method for low-molecular weight chondroitin sulfate, described production method comprises: use the chondroitin sulfate A (CSA) BC enzyme mutant fusion rotein described in any one of claim 1 ~ 4 to produce low-molecular weight chondroitin sulfate.
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| CN105441471A (en) * | 2015-10-28 | 2016-03-30 | 北京电子科技职业学院 | Preparation method and application of chondroitin sulfate ABC enzyme fusion protein |
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| US9796970B1 (en) | 2017-04-24 | 2017-10-24 | Advantek Serum Laboratories Ltd. | Production of high purity chondroitinase ABC |
| CN109897812A (en) * | 2019-03-19 | 2019-06-18 | 江南大学 | It is a kind of express chondroitin 4- sulfotransferase gene recombinant bacterium and its application |
| CN109897812B (en) * | 2019-03-19 | 2021-09-03 | 江南大学 | Recombinant bacterium for expressing chondroitin 4-sulfate transferase gene and application thereof |
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| CN111518822B (en) * | 2019-07-02 | 2022-08-09 | 江南大学 | Chondroitin sulfate ABC lyase mutant and secretory expression method thereof |
| CN110777140B (en) * | 2019-12-04 | 2021-07-06 | 北京电子科技职业学院 | Chondrosulphatase AC mutant and preparation method thereof |
| CN112877316A (en) * | 2021-01-25 | 2021-06-01 | 南京汉欣医药科技有限公司 | Novel chondroitinase sulfate and lactococcus lactis expressing chondroitinase sulfate |
| CN113461796A (en) * | 2021-08-04 | 2021-10-01 | 深圳晶蛋生物医药科技有限公司 | Recombinant SGLT2 protein and application thereof |
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