CN103966252A - Application of cellulose binding domain in secretory expression system of corynebacteria - Google Patents
Application of cellulose binding domain in secretory expression system of corynebacteria Download PDFInfo
- Publication number
- CN103966252A CN103966252A CN201310045414.1A CN201310045414A CN103966252A CN 103966252 A CN103966252 A CN 103966252A CN 201310045414 A CN201310045414 A CN 201310045414A CN 103966252 A CN103966252 A CN 103966252A
- Authority
- CN
- China
- Prior art keywords
- cbd
- protein
- cellulose
- sequence
- recombinant
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Abstract
The invention specifically relates to a method of using the cellulose binding domain of Trichoderma reesei as an affinity tag for secretory expression and purification of recombinant protein in a corynebacteria expression system. The method comprises the following steps: 1) optimizing and synthesizing a codon sequence coding the cellulose binding domain of Trichoderma reesei glucoside hydrolase I; 2) cloning the sequence synthesized in the step 1 to a corynebacteria secretory expression vector as a protein tag sequence; 3) inserting a target protein sequence into the tag vector obtained in the step 2; 4) transforming the recombinant vector obtained in the step 3 to corynebacteria for secretory expression of protein; and 5) with CBD as the affinity tag, purifying recombinant protein secreted by the recombinant bacterium by using a cellulose column. According to the method, fragmentation of thalli is not needed, a low-cost cellulose column filling material is directly used for protein purification of an induced medium, so production cost for protein is reduced.
Description
Technical field
The invention belongs to protein expression and purification technology field, be specially and utilize cellulose binding domain as affinity tag, the method for the recombinant protein of fast purifying coryneform bacteria secreting, expressing.The present invention relates to the structure of the coryneform bacteria secretion expression carrier taking CBD as affinity tag, and utilize the secreted recombinant protein of cellulose column purifying coryneform bacteria.
Background technology
The pathogenic gram-positive microorganism of coryneform bacteria right and wrong, the amino acid and the VITAMIN that are often used to produce extensive stock
[1-4].Through mutation breeding for many years, the improvement of protoplastis and expression vector, is expected to for large-scale protein production
[5,6].2006, the people such as the Date of Japan utilized corynebacterium glutamicum secreting, expressing in substratum to provide activated human epidermal growth factor, illustrate that this system has the ability of scale operation activated protein
[7].There is extremely significantly advantage using coryneform bacteria as expressive host: 1) genetic background is clear, has efficient secreting signal peptide and molecular chaperones system, and secreted recombinant protein has native conformation and biological activity mostly
[5]; 2) there is the approach of two kinds of secretory proteins: except conventional Secretory Pathway, also can pass through double arginine transposition (Twin-arginine translocation, Tat) approach and secrete, make expressed albumen direct secretion in substratum, simplify purification step, reduce production costs
[8]; 3) belong to gram-positive microorganism, do not produce the intracellular toxins such as lipopolysaccharides, security is good; 4) proteolytic enzyme of secretion is few, can impact expressed albumen hardly, and product is stable
[5]; 5) of long duration as expressive host, people have accumulated extremely abundant fermentation experience, strictly aerobic, and rapidly, culture condition is simple in growth, uses safety, and no pathogenicity, so corynebacterium glutamicum is a kind of very good engineering strain.
CBD (Cellulose-binding domain, CBD) is carbohydrate inversion enzyme---the albumen territory of a cellulose-binding of cellulase, can improve the cellulase activity of bacterium or fungi
[9,10].There are several hydrophobic surfaces that contains die aromatischen Aminosaeuren residue in this albumen territory, can affinely be adsorbed onto the hydrophobic region of cellulose surface
[11-13].Utilizing affinity tag purification of recombinant proteins is the technological method being in daily use in biotechnology, and conventional label has poly Histidine, glutathione sulfydryl transferase, maltose binding protein, streptavidin, chitin binding domain or their aggregate etc.
[14,15].Can be also one of them with the CBD of Mierocrystalline cellulose generation reversible adsorption, comparatively independently space structure be less to the disturbing influence of protein biology performance for it, and Mierocrystalline cellulose can significantly reduce separation costs as absorption raw material
[16].In recent years it is that purification tag carries out protein purification that some are studied successful Application CBD
[17-19].
In the expression system of corynebacterium glutamicum, being directly secreted into target protein outside born of the same parents can be by centrifugal, dialysis, and the methods such as concentrated or affinity chromatography obtain.The high manufacturing cost of the damping fluid that these methods are used and affine resin can strengthen the cost of protein purification, and in large-scale production application, the important factor that cost must be considered often.This research application CBD label is optimized the purifying process of corynebacterium glutamicum expression system, attempts to reduce the protein purification cost of this system.
Summary of the invention
The present invention is a kind of purification process of secreting, expressing recombinant protein, the specifically purification tag taking CBD as coryneform bacteria secreting, expressing system, utilize Microcrystalline Cellulose as column chromatography filler, the recombinant protein of coryneform bacteria secretion to be carried out the method for purifying, the purifying process that is intended to optimize protein expression, reduces costs.Relate to containing the structure of the coryneform bacteria secretion expression carrier of affinity tag CBD with cellulose wadding the recombinant protein of secretion is carried out to purifying.
The invention provides following:
1, utilize the cellulose binding domain of Trichodermareesei as affinity tag, in coryneform expression system, carry out the method for secreting, expressing and purification of recombinant proteins.Said method comprising the steps of:
1) optimize the also DNA sequence dna of the cellulose binding domain (CBD) of composite coding Trichodermareesei glycoside hydrolase I;
2) sequence synthetic step 1 is cloned into coryneform bacteria secretion expression carrier as albumen sequence label;
3) target protein sequence is inserted into the label carrier that step 2 obtains;
4) recombinant vectors step 3 being obtained is converted into the secreting, expressing that carries out albumen in coryneform bacteria;
5), taking CBD as affinity tag, utilize the recombinant protein of this recombinant bacterium secretion of cellulose column purifying.
2, in the method for claim 1, step 1) cellulose binding domain CBD derive from CBD and the connection peptides thereof of Trichodermareesei glycoside hydrolase I, its aminoacid sequence is SEQ ID NO:1.Wherein the aminoacid sequence of CBD is SEQ ID NO:2, and the aminoacid sequence of connection peptides is SEQ ID NO:3.
3, in the method for claim 1, step 2) expression vector be coryneform secretion expression carrier, its secretion signal element is the signal peptide cgR-sp of the cgR albumen in corynebacterium glutamicum source, its aminoacid sequence is SEQ ID NO:4.
4, in claim 1, step 3) described target protein can be inserted into the N end of CBD label, also can be inserted into the C end of CBD label.
5, in the method for claim 1, step 4) conversion host be coryneform bacteria, comprise corynebacterium glutamicum (Corynebacterium glutamicum), microtissue coryneform bacteria (Corynebacteriumcallunae), have a liking for etheric acid coryneform bacteria (Corynebacterium acetoacidophilum).
6, in the method for claim 1, step 5) in comprise Microcrystalline Cellulose for the cellulose wadding of protein purification, amorphous cellulose.
First aspect involved in the present invention---build a coryneform bacteria secretion expression carrier containing CBD affinity tag, described CBD label derives from the Trichodermareesei glycoside hydrolase I stronger than enzyme, consider labeling requirement comparatively independently space structure reduce the impact of the biological property on target protein, the present invention is optimizing and comprise its part connection peptides sequence when synthetic CBD sequence, its aminoacid sequence is SEQID NO:1, and after optimizing, synthetic DNA sequence dna is SEQ ID5.The secretion signal element of described secretion expression carrier is the selection result with reference to corynebacterium glutamicum signal peptide, selects the signal peptide cgR-sp of the cgR albumen (GI:145295004) in corynebacterium glutamicum source
[20], its aminoacid sequence is SEQ IDNO:4.
Second aspect involved in the present invention---with cellulose wadding to secretion restructuring label protein carry out purifying.The present invention taking corynebacterium glutamicum as host sets forth method of the present invention, but described method also comprises the microtissue coryneform bacteria that belongs to equally coryneform bacteria and have efficient secretory expression ability, has a liking for etheric acid coryneform bacteria.Described purification process first Application cellulose column is discharged into the cbd fusion protein of cultivation during reversibly in conjunction with coryneform bacteria secreting, expressing, thereby does not need to carry out bacterial cell disruption and obtain target protein.
Below concrete operations of the present invention:
1) first to carry out the codon optimized of coryneform bacteria expression to the CBD of Trichodermareesei cellobiohydrolase I and connection peptides thereof, thereby improve the expression efficiency of CBD label in coryneform bacteria.
Method: utilize conventional codon optimized software to carry out after prokaryotic expression codon optimized CBD and connection peptides thereof, delivering to gene Synesis Company, to carry out DNA sequence dna synthetic.
2) sequence of CBD after optimizing is inserted into the coryneform bacteria secretion expression carrier using cgR-sp as signal peptide, builds the corynebacterium glutamicum secretion expression carrier taking CBD as purification tag.
Method: the molecular cloning method connecting by conventional restriction enzyme digestion.
3) label carrier target protein sequence clone to step 2 being obtained.
Method: the molecular cloning method connecting by conventional restriction enzyme digestion.
4) recombinant vectors step 3 being obtained is converted into and in coryneform bacteria, carries out secreting, expressing.
Method: after transforming by electricity, the method for heat shock is converted into corynebacterium glutamicum by constructed plasmid and induces also secreting, expressing.
5), taking CBD as purification tag, utilize the recombinant protein of this recombinant bacterium secretion of cellulose column purifying.
Method: concrete grammar is with reference to following embodiment.
Brief description of the drawings:
Fig. 1: build the expression of recombinant proteins purification system taking CBD as purification tag in corynebacterium glutamicum.
Fig. 2: the amalgamation mode of recombinant protein.
Fig. 3: the secreting, expressing situation of recombinant protein in substratum.The thick liquid of albumen under A white light, the thick liquid of albumen under B UV-light;
Fig. 4: the SDS-PAGE of the thick liquid of albumen analyzes.
Fig. 5: the SDS-PAGE of protein purification gained sample.A cellulose column purifying, B ni-sepharose purification.
Fig. 6: the cellulose column in purge process.
1C.glutamicum-pXMJ19-sp，2C.glutamicum-pXMJ19-sp-GFP-His，3C.glutamicum-pXMJ19-sp-GFP-CBD
The secreting, expressing of embodiment: eGFP-CBD and cellulose column purifying thereof
EGFP is a kind of green fluorescent protein, is usually used in molecule location and protein labeling.The present embodiment is taking eGFP as example, by it in corynebacterium glutamicum with fusion secreting, expressing and the purifying of CBD, thereby show the application of CBD in affinity chromatography.
Bacterial classification and reagent
Intestinal bacteria E.coli DH5a, plasmid pMXS-IRES-GFP, corynebacterium glutamicum C.glutamicum13032, corynebacterium glutamicum expression vector pXMJ19-sp are for having by oneself in this laboratory, and DNA purifying reclaims test kit, plasmid extraction kit, albumen Marker purchased from the biological company limited of sky root; T4 ligase enzyme, Taq polysaccharase and various restriction enzyme are purchased from NEB company; Nickel post, dialysis membrane are purchased from Sheng Gong biotechnology company limited; Microcrystalline Cellulose filler Avicel PH-102NF (lot number: P210822506) is purchased from FMC Biopolymer; BCA determination of protein concentration test kit is purchased from green skies biotechnology research institute; All the other reagent are domestic or Import Analysis is pure.LB substratum (for the cultivation of intestinal bacteria and corynebacterium glutamicum): 0.5%Yeast extract, 1%Tryptone, 1%NaCl, 5 μ g/mL paraxin; MMTG substratum (for the fermentation culture of corynebacterium glutamicum, can promote secreting, expressing): 6% glucose, 0.1%MgSO
4, 3% (NH
4)
2sO
4, 0.15%KH
2pO
4, 0.001%FeSO
4.7H
2o, 0.001%MnSO
4.4H
2o, 450 μ g/L Thiamine hydrochloride, VB1,450 μ g/L vitamin Hs, 0.015% methionine(Met), 5%CaCO
3, pH=7.5
[21].
1) structure of eGFP-CBD secretion expression carrier
Gene is synthetic: pass through
http:// www.jcat.de/Result.jspthe CBD of glycoside hydrolase I etc. web site software to Trichodermareesei and connection peptides thereof are carried out the codon optimized of corynebacterium glutamicum expression, and the DNA sequence dna after optimization is SEQ ID NO:5.After AgeI and PstI restriction enzyme site are added respectively in two ends, delivering to biotech firm, to carry out DNA synthetic.
Label carrier builds: the DNA sequence dna after synthetic is cut and is connected to corynebacterium glutamicum secretion expression carrier pXMJ19-sp by AgeI and PstI enzyme, build the CBD label carrier pXMJ19-sp-CBD of corynebacterium glutamicum secreting, expressing.Wherein the signal peptide sequence of pXMJ19-sp is SEQ ID NO:4.
Protein expression vector builds: taking pMXS-IRES-GFP as template, pcr amplification two ends are added Xhol and PstI restriction enzyme site and do not contained the gene fragment of the eGFP of terminator, and the primer using is SEQ ID:6 and SEQ ID:7.PCR reaction conditions is: 94 DEG C of sex change 4min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C are extended 45s, circulate 30 times; 72 DEG C are extended 10min.Then use Xhol and AgeI double digestion gained PCR product and label carrier pXMJ19-sp-CBD, the fragment of connection after glue reclaims builds the eGFP secretion expression carrier pXMJ19-sp-eGFP-CBD of the corynebacterium glutamicum taking CBD as purification tag.
2) secreting, expressing of recombinant protein
With reference to the people's such as Van method, after above-mentioned carrier is transformed by electricity, constructed plasmid is converted into corynebacterium glutamicum C.glutamicum13032 by the method for heat shock
[22]in, thereby build restructuring corynebacterium glutamicum C.glutamicum-pXMJ19-sp-GFP-CBD.The recombinant bacterium of overnight incubation is inoculated in 100mL MMTG substratum in 1: 100 ratio, 30 DEG C, 200rpm, being cultured to bacterium liquid OD value is 0.4～0.6.Adding final concentration is that the IPTG of 1mmol/L cultivates 3 days in 30 DEG C, can induce the secreting, expressing of target protein.By above-mentioned zymocyte liquid at 4 DEG C, the centrifugal 10min of 8000r/min, gained supernatant substratum is the thick liquid of albumen, under the irradiation of 100W ultraviolet lamp, seeing through yellow-green colour filter takes pictures to the thick liquid of albumen, the fluorescence situation of record as shown in Figure 3, the thick liquid of albumen is aobvious green, illustrate that this recombinant bacterium secretes recombinant protein GFP-CBD to substratum, make it send green fluorescence at ultraviolet lamp.As shown in Figure 4, this recombinant bacterium is secreted into the size of the recombinant protein GFP-CBD in substratum at 33KDa to 15%SDS polyacrylamide (SDS-PAGE) the electrophoretic analysis result of the thick liquid of albumen, meets expection size.
3) purifying of recombinant protein
Add the ammonium sulfate of final concentration 1mol/L in the thick liquid of above-mentioned albumen after, upper prop is to the 1.5ml cellulose column through 1mol/L ammonium sulfate balance, clean 10ml and 3ml with 1mol/L ammoniumsulphate soln and 0.5mol/L ammoniumsulphate soln respectively, finally use distilled water wash-out 5ml, collect the result of each step liquid analyzing proteins purifying in 15%SDS polyacrylamide (SDS-PAGE) electrophoresis as shown in Figure 5, illustrate that utilizing cheaply cellulose wadding can purifying to obtain this recombinant bacterium is secreted into the recombinant protein in substratum.
The secreting, expressing of comparative example 1:eGFP-His and ni-sepharose purification thereof
EGFP brief introduction, bacterial classification, reagent etc. refer to embodiment
1) structure of eGFP-His secretion expression carrier:
Taking pMXS-IRES-GFP as template, therefrom increase C end with the GFP-His gene fragment of His label and at two ends interpolation Xhol and PstI restriction enzyme site by PCR, the primer using is SEQID:6 and SEQ ID:8.PCR reaction conditions is: 94 DEG C of sex change 4min; 94 DEG C of sex change 30s, 55 DEG C of annealing 30s, 72 DEG C are extended 45s, circulate 30 times; 72 DEG C are extended 10min.Then use Xhol and PstI double digestion GFP-His gene fragment and expression vector pXMJ19-sp, the fragment of connection after glue reclaims builds the GFP expression vector pXMJ19-sp-GFP-His of the corynebacterium glutamicum taking poly Histidine as purification tag.
2) secreting, expressing of recombinant protein
After above-mentioned carrier is transformed by electricity, constructed plasmid is converted into corynebacterium glutamicum C.glutamicum13032 by the method for heat shock
[22]middle structure restructuring corynebacterium glutamicum C.glutamicum-pXMJ19-sp-GFP-His.The recombinant bacterium of overnight incubation is inoculated in 100mL MMTG substratum in 1: 100 ratio, 30 DEG C, 200rpm, being cultured to bacterium liquid OD value is 0.4～0.6.Adding final concentration is that the IPTG of 1mmol/L cultivates 3 days in 30 DEG C, can induce the secreting, expressing of target protein.By above-mentioned zymocyte liquid at 4 DEG C, the centrifugal 10min of 8000r/min, gained supernatant substratum is the thick liquid of albumen, under the irradiation of 100W ultraviolet lamp, seeing through yellow-green colour filter takes pictures to the thick liquid of albumen, the fluorescence situation of record as shown in Figure 3, the thick liquid of albumen is aobvious green, illustrate that this recombinant bacterium secretes recombinant protein GFP-His to substratum, make it show green fluorescence at ultraviolet lamp.In 15%SDS polyacrylamide (SDS-PAGE) electrophoretic analysis result as shown in Figure 4, this recombinant bacterium is secreted into recombinant protein GFP-CBD in substratum at 28KD to the thick liquid of albumen, meets expection size.
3) purifying of recombinant protein
Add final concentration 400mmol/L NaCl in the thick liquid of the above-mentioned albumen of 30ml after, upper prop is to the nickel post of 1.5ml through phosphoric acid buffer pre-equilibration, clean 10ml and 3ml with the phosphoric acid buffer containing 10mmol/L imidazoles with containing the phosphoric acid buffer of 20mmol/L imidazoles respectively, finally, with the phosphoric acid buffer wash-out 5ml containing 250mmol/L imidazoles, collect each step liquid.Collect each step liquid in the result of 15%SDS polyacrylamide (SDS-PAGE) electrophoretic analysis protein purification as shown in Figure 5, illustrate and utilize traditional nickel column packing to be secreted into the recombinant protein in substratum by purification of Recombinant bacterium.。
Comparative example 2: the secreting, expressing analysis of empty carrier recombinant bacterium
1) construction and expression of empty carrier recombinant bacterium
After pXMJ19-sp is transformed by electricity, constructed plasmid is converted into corynebacterium glutamicum C.glutamicum13032 by the method for heat shock
[22]middle structure restructuring corynebacterium glutamicum C.glutamicum-pXMJ19-sp.The recombinant bacterium of overnight incubation is inoculated in 100mLMMTG substratum in 1: 100 ratio, 30 DEG C, 200rpm, being cultured to bacterium liquid OD value is 0.4～0.6.Adding final concentration is that the IPTG of 1mmol/L cultivates 3 days in 30 DEG C, can induce the secreting, expressing of target protein.By above-mentioned zymocyte liquid at 4 DEG C, the centrifugal 10min of 8000r/min, gained supernatant substratum is the thick liquid of albumen, under the irradiation of 100W ultraviolet lamp, see through yellow-green colour filter the thick liquid of albumen is carried out to Taking Pictures recording, fluorescence intensity as shown in Figure 1, the thick liquid of albumen is not aobvious green, illustrates containing the recombinant bacterium of empty carrier and can not secrete target protein.In 15%SDS polyacrylamide (SDS-PAGE) electrophoretic analysis result as shown in Figure 4, in the thick liquid of albumen, without target protein, illustrate containing the recombinant bacterium of empty carrier and can not secrete target protein.
The primer using in table 1 embodiment
Experimental result
Each recombinant bacterium (C.glutamicum-pXMJ19-sp-GFP-CBD of secreting, expressing effect: embodiment, comparative example 1 and comparative example 2, C.glutamicum-pXMJ19-sp-GFP-His, C.glutamicum-pXMJ19-sp) substratum after secretion inducing expression as shown in Figure 3: under ultraviolet lamp, the above two show strong green fluorescence, and the latter does not have; Three produce the thick liquid of albumen by PEG dialyse concentrate after carry out SDS-PAGE analysis, result is as shown in Figure 4: C.glutamicum-pXMJ19-sp-GFP-His and the about 30KDa of C.glutamicum-pXMJ19-sp-GFP-CBD institute abduction delivering target protein molecular weight, this is consistent with calculated value (28KDa and 33KDa), detect through BCA protein concentration detection kit, can estimate that the recombinant protein secreting, expressing amount of this system can reach 200mg/ml.
The thick liquid of albumen of purification effect: embodiment and contrast enforcement 1 carries out respectively cellulose column purifying and ni-sepharose purification, get the thick liquid of gained albumen, stream wears liquid and elutriant carries out SDS-PAGE analysis, ni-sepharose purification result as shown in Figure 5 B, by the pure target protein that obtains 90% above purity of nickel post, and cellulose column purification result as shown in Figure 5A: also can obtain the target protein of 90% above purity by cellulose column purifying, illustrate that purification effect of the present invention is suitable with nickel post.Wherein in embodiment, Microcrystalline Cellulose price used is 3 yuan/ml, and in comparative example 1, nickel column packing price used is 50 yuan/ml.
In sum, the experimental result of secreting, expressing effect shows that coryneform bacteria expression system of the present invention can carry out expression-secretion by add signal peptide before recombinant protein, thereby without bacterial cell disruption, obtains crude protein liquid, reduces costs; The experimental result of purification effect shows can directly carry out purifying recovery to the CBD recombinant protein in substratum by cellulose column, reaches the effect identical with traditional nickel column purification with lower cost, further reduces costs.
Reference
[1]
R.Secretion of amino acids by bacteria：Physiology and mechanism[J].FEMS Microbiology Reviews，1994，13(1)：75-93.
[2]Hermann T.Industrial production of amino acids by coryneform bacteria[J].JBiotechnol，2003，104(1-3)：155-172.
[3]Huser A T，Chassagnole C，Lindley N D，etal.Rational design of aCorynebacterium glutamicum pantothenate production strain and itscharacterization by metabolic flux analysis and genome-wide transcriptionalprofiling[J].Appl Environ Microbiol，2005，71(6)：3255-3268.
[4] also know quiet Lee, Shi Jing, Wang little Yuan. the kinase whose mistake of Corynebacterium glutamicum NAD expressed the promoter action [J] synthetic to ILE. biotechnology journal, 2012,28 (9): 1038～1047.
[5]Lothar Eggeling M B.Handbook of Corynebacterim glutamicum[J].CaisterAcademic Press，Boca Rotan，2005，
[6]Burkovski.Corynebacteria：Genomics and Molecular Biology[J].CaisterAcademic Press，Norfolk.，2008，
[7]Date M，Itaya H，Matsui H，et al.Secretion of human epidermal growth factorby Corynebacterium glutamicum[J].Lett Appl Microbiol，2006，42(1)：66-70.
[8]Meissner D，Vollstedt A，van Dijl J M，et al.Comparative analysis oftwin-arginine(Tat)-dependent protein secretion of a heterologous model protein(GFP)in three different Gram-positive bacteria[J].Appl Microbiol Biotechnol，2007，76(3)：633-642.
[9]TommeP，Van Tilbeurgh H，Pettersson G，et al.Studies of the cellulolyticsystem of Trichoderma reesei QM9414.Analysis of domain function in twocellobiohydrolases by limited proteolysis[J].Eur J Biochem，1988，170(3)：575-581.
[10]Gilkes N R，Warren R A，Miller R C，Jr.，et al.Precise excision of thecellulose binding domains from two Cellulomonas fimi cellulases by ahomologous protease and the effect on catalysis[J].J Biol Chem，1988，263(21)：10401-10407.
[11]Lehtio J，Sugiyama J，Gustavsson M，et al.The binding specificity andaffinity determinants of family1and family3cellulose binding modules[J].ProcNatl Acad Sci U S A，2003，100(2)：484-489.
[12]Ding S Y，Xu Q，Ali M K，et al.Versatile derivatives of carbohydrate-bindingmodules for imaging of complex carbohydrates approaching the molecular levelof resolution[J].Biotechniques，2006，41(4)：435-436，438，440passim.
[13]Dagel D J，Liu Y S，Zhong L，et al.In situ imaging of singlecarbohydrate-binding modules on cellulose microfibrils[J].J Phys Chem B，115(4)：635-641.
[14]Mateo C，Fernandez-Lorente G，Pessela B C，et al.Affinity chromatographyof po1yhistidine tagged enzymes.New dextran-coated immobilized metal ionaffinity chromatography matrices for prevention of undesired multipointadsorptions[J].J Chromatogr A，2001，915(1-2)：97-106.
[15]Sakhamuru K，Hough D W，Chaudhuri J B.Protein purification byultrafiltration using a beta-galactosidase fusion tag[J].Biotechnol Prog，2000，16(2)：296-298.
[16]Shoseyov O，Shani Z，Levy I.Carbohydrate binding modules：biochemicalproperties and novel applications[J].Microbiol Mol Biol Rev，2006，70(2)：283-295.
[17]Kavoosi M，Meijer J，Kwan E，et al.Inexpensive one-step purification ofpolypeptides expressed in Escherichia coli as fusions with the family9carbohydrate-binding module of xylanase10A from T.maritima[J].J ChromatogrB Analyt Technol Biomed LifeSci，2004，807(1)：87-94.
[18]Hilden L，Johansson G.Recent developments on cellulases andcarbohydrate-binding modules with cellulose affinity[J].Biotechnol Lett，2004，26(22)：1683-1693.
[19] as if big vast Beamy, Gao Xiaolian. family's 3 cellulose binding domains in eukaryote as the application [J] of the affinity tag of recombinant protein expression and purifying. China, 2011, (201110035017):
[20]Watanabe K，Tsuchida Y，Okibe N，et al.Scanning the Corynebacteriumglutamicum R genome for high-efficiency secretion signal sequences[J].Microbiology，2009，155(Pt3)：741-750.
[21]Date M，Yokoyama K，Umezawa Y，et al.High level expression ofStreptomyces mobaraensis transglutaminase in Corynebacterium glutamicumusing a chimeric pro-region from Streptomyces cinnamoneus transglutaminase[J].J Biotechnol，2004，110(3)：219-226.
[22]van der Rest M E，Lange C，Molenaar D.A heat shock followingelectroporation induces highly efficient transformation of Corynebacteriumglutamicum with xenogeneic plasmid DNA[J].Appl Microbiol Biotechnol，1999，52(4)：541-545.
Claims (6)
1. utilize the cellulose binding domain of Trichodermareesei to express in coryneform secreting, expressing system as affinity tag and the method for purification of recombinant proteins, said method comprising the steps of:
1) optimize the also DNA sequence dna of the cellulose binding domain (CBD) of composite coding Trichodermareesei glycoside hydrolase I;
2) sequence synthetic step 1 is cloned into coryneform bacteria secretion expression carrier as albumen sequence label;
3) target protein sequence is inserted into the label carrier that step 2 obtains;
4) recombinant vectors step 3 being obtained is converted into the secreting, expressing that carries out albumen in coryneform bacteria;
5), taking CBD as affinity tag, utilize the recombinant protein of this recombinant bacterium secretion of cellulose column purifying.
2. in the method for claim 1, step 1) cellulose binding domain CBD derive from CBD and the connection peptides thereof of Trichodermareesei glycoside hydrolase I, its aminoacid sequence is SEQ ID NO:1.Wherein the aminoacid sequence of CBD is SEQ ID NO:2, and the aminoacid sequence of connection peptides is SEQ ID NO:3.
3. in the method for claim 1, step 2) expression vector be coryneform secretion expression carrier, its secretion signal element is the signal peptide of the cgR albumen in corynebacterium glutamicum source, its aminoacid sequence is SEQ ID NO:4.
4. in claim 1, step 3) described target protein sequence can be inserted into the N end of CBD label, also can be inserted into the C end of CBD label.
5. in the method for claim 1, step 4) conversion host be coryneform bacteria, comprise corynebacterium glutamicum (Corynebacterium glutamicum), microtissue coryneform bacteria (Corynebacteriumcallunae), have a liking for etheric acid coryneform bacteria (Corynebacterium acetoacidophilum).
6. in the method for claim 1, step 5) in comprise Microcrystalline Cellulose for the cellulose wadding of protein purification, amorphous cellulose.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310045414.1A CN103966252A (en) | 2013-01-28 | 2013-01-28 | Application of cellulose binding domain in secretory expression system of corynebacteria |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201310045414.1A CN103966252A (en) | 2013-01-28 | 2013-01-28 | Application of cellulose binding domain in secretory expression system of corynebacteria |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN103966252A true CN103966252A (en) | 2014-08-06 |
Family
ID=51236256
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201310045414.1A Pending CN103966252A (en) | 2013-01-28 | 2013-01-28 | Application of cellulose binding domain in secretory expression system of corynebacteria |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN103966252A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10081802B2 (en) | 2013-07-29 | 2018-09-25 | Danisco Us Inc. | Variant Enzymes |
| CN112608932A (en) * | 2020-12-02 | 2021-04-06 | 华农（肇庆）生物产业技术研究院有限公司 | Method for efficiently expressing avian adenovirus Fiber-2 protein in escherichia coli |
-
2013
- 2013-01-28 CN CN201310045414.1A patent/CN103966252A/en active Pending
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US10081802B2 (en) | 2013-07-29 | 2018-09-25 | Danisco Us Inc. | Variant Enzymes |
| US10479983B2 (en) | 2013-07-29 | 2019-11-19 | Danisco Us Inc | Variant enzymes |
| CN112608932A (en) * | 2020-12-02 | 2021-04-06 | 华农（肇庆）生物产业技术研究院有限公司 | Method for efficiently expressing avian adenovirus Fiber-2 protein in escherichia coli |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Blaak et al. | Binding and substrate specificities of a Streptomyces olivaceoviridis chitinase in comparison with its proteolytically processed form | |
| CN105755023A (en) | ZEN (zearalenone) degrading enzyme gene and high-yield strain | |
| CN105985968A (en) | Improved broad-spectrum endonuclease and industrial production method thereof | |
| CN109439642A (en) | 61 protein gene of glycoside hydrolase Families and its albumen and preparation method | |
| CN105296506B (en) | Target protein expression and purification method using laetiporus sulphureus mushroom lectin N-acetamido lactosamine binding domain as fusion tag | |
| CN105481954A (en) | Recombinant protein A and applications thereof | |
| CN102140476A (en) | Gene of recombinant staphylococal protein A, expression vector containing gene and application thereof | |
| CN103966252A (en) | Application of cellulose binding domain in secretory expression system of corynebacteria | |
| CN102174532A (en) | Application of family 3 cellulose binding domain serving as affinity tag for expression and purification of recombinant protein in eukaryote | |
| US6407208B1 (en) | Chimeric proteins with a cellulose binding domain | |
| CN103184229A (en) | Recombinant protein A/G gene and preparation and application of expression product of recombinant protein A/G gene | |
| CN102260694A (en) | Acidproof medium-temperature alpha-amylase and preparation method thereof | |
| CN105950596B (en) | A kind of difunctional acid urease gene and its expression and application | |
| WO2023221787A1 (en) | Pichia pastoris engineering strain for recombinant type i collagen, construction method therefor and use thereof | |
| CN107119058A (en) | A kind of MS2 capsid proteins fusion expression method and its application | |
| JP6350987B2 (en) | Thermostable β-xylosidase belonging to GH family 3 | |
| CN106884030A (en) | The method for improving the fermentation yield of the fusion protein of containing cellulose binding domain | |
| CN103320413B (en) | Aspergillus-versicolor-derived alpha-galactosidase Z-GALC, and coding gene and application thereof | |
| CN106754848B (en) | Alkaline pectinase mutant with improved thermal stability | |
| CN104140972B (en) | The preparation method of diphtheria toxin muton CRM 197 | |
| JP6364662B2 (en) | Thermostable β-xylosidase belonging to GH family 3 | |
| CN103305522B (en) | A kind of nucleotide sequence of optimum combination Human Ciliary Neurotrophic Factor and in intestinal bacteria solution expression with high efficiency method | |
| Yaron et al. | Expression, purification and crystallization of a cohesin domain from the cellulosome of Clostridium thermocellum | |
| CN109182361A (en) | Cellulose incision enzyme gene and its albumen and application | |
| CN103320414B (en) | Neutral alpha-galactosidase M-GALC, and coding gene and application thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C02 | Deemed withdrawal of patent application after publication (patent law 2001) | ||
| WD01 | Invention patent application deemed withdrawn after publication |
Application publication date: 20140806 |