CN105481954A - Recombinant protein A and applications thereof - Google Patents
Recombinant protein A and applications thereof Download PDFInfo
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- CN105481954A CN105481954A CN201410471277.2A CN201410471277A CN105481954A CN 105481954 A CN105481954 A CN 105481954A CN 201410471277 A CN201410471277 A CN 201410471277A CN 105481954 A CN105481954 A CN 105481954A
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Abstract
The present invention relates to a recombinant protein A and applications thereof, and particularly discloses a novel recombinant Staphylococcus aureus protein A, which has a structure defined in the specification, wherein the function comprises but is not limited to application as a ligand-specific binding immunoglobulin, and the recombinant protein A can be used as a specific affinity ligand, and has excellent alkali resistance. The invention further relates to a method for constructing the mutant protein.
Description
Technical field
The invention belongs to biological technical field.Particularly, the present invention relates to a kind of novel restructuring staphylococcus aureus protein A and the application in separation and purification of protein thereof.
Background technology
Staphylococcus aureus protein A (StaphylococalProteinA, SpA) comprises 5 structural domains, is respectively E, D, A, B, C, and wherein B structural domain (B fragment) is the strongest to binding domain-immunoglobulin bonding force.Wild-type SP and Recombinant Staphylococal Protein A (rPA) and modified form albumen are widely used in field of affinity chromatography, the affinity chromatography medium be made up of SP is widely used in capture antibody, removes host protein and residual DNA.
Affinity chromatography medium often need cleaning, object be for retain affinity ligand selectivity and high degree of specificity and prevent bacterial contamination, usually adopt 0.1 ~ 0.5MNaOH solution to rinse, its pH scope is between 12 ~ 14.Because albumen easily sex change occurs under this alkaline condition, lose activity function.Therefore, affinity chromatography medium is carried out alkali lye situ cleaning, repeatedly after circulation, medium will significantly be lost the bonding force of target molecule, not only increase the weight of purifying cost, and, due to coming off of albumin A, also reduce the purity of target product.
The B fragment of staphylococcus aureus protein A is through being transformed into Z fragment, the glycine that its aminoacid sequence is the 29th is replaced by L-Ala (Stahl etc., 1999.Affinityfusionsinbiotechnology:focusonproteinAandpr oteinG, inTheEncyclopediaofBioprocessTechnology:Fermentation, BiocatalysisandBioseparation.M.C.Fleckinger and S.W.Drew, editor .JohnWileyandSonsInc., New York, 8-22).Z fragment is used as the aglucon in affinity chromatography, compared to B fragment, Z fragment demonstrates the stability strengthened some chemical reagent except NaOH, but under higher alkaline condition, remain unstable, the needs that industrial on-line cleaning (CleaningInPlace, CIP) regenerates can not be satisfied with.Therefore, this area still needs exploitation one more stable, the albumin A that especially alkali resistance is stronger.
Summary of the invention
The object of this invention is to provide a kind of novel restructuring staphylococcus aureus protein A aggressiveness, its function includes but not limited to as a kind of aglucon binding domain-immunoglobulin specifically.
In a first aspect of the present invention, provide a kind of recombinant protein A aggressiveness, described recombinant protein A aggressiveness is the polymer that the Z fragment series connection of two or more staphylococcus aureus protein A is formed, and at least one in each Z fragment is the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
In another preference, each in described Z fragment is the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
In another preference, described recombinant protein A aggressiveness is 2-20 aggressiveness, preferably 4-10 aggressiveness.
In another preference, described recombinant protein A aggressiveness is 4 aggressiveness, 6 aggressiveness, 8 aggressiveness, 10 aggressiveness.
In another preference, two Z fragments adjacent in described recombinant protein A aggressiveness are directly connected, or are connected by connection peptides.
In another preference, described recombinant protein A aggressiveness is made up of Z fragment and connection peptides; Described connection peptides is present between Z fragment, and described Z fragment is all the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
In another preference, described recombinant protein A aggressiveness has the structure shown in formula (I):
Pa-(Za-L)n-Za-Pb(I)
In formula,
Pa be nothing, Met, leader sequence, secretion peptide sequence, for the sequence label of purifying or its combination;
Each Za is the aminoacid sequence of the Z fragment of staphylococcus aureus protein A independently, and at least one Za is the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr;
L is nothing or connection peptides sequence independently;
N is the positive integer of >=1;
Pb is nothing, Cys, the sequence label of purifying or its combination.
In another preference, n is the positive integer of 1-50, preferably the positive integer of 2-20, preferably 2,3,4,5,6,7 or 8.
In another preference, all Za are the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
In another preference, the sequence of described Za is as shown in SEQIDNo.:3.
In another preference, described L is length 3-50 (preferably 3-20) amino acid whose connection peptides sequence.
In another preference, described L is selected from lower group: QKDAVFP, ASTKGP.
In another preference, described recombinant protein A aggressiveness has structure shown in formula (II):
Met-Za-L-Za-L-Za-L-Za-Cys(II)
Wherein, Za, L definition is the same.
In another preference, described recombinant protein A aggressiveness is the albumen of tool antibody binding capacity.
In another preference, described antibody comprises IgG antibody, IgM antibody, IgA antibody.
In another preference, the C-terminal of described recombinant protein A aggressiveness is also containing amino-acid residue Cys.
In another preference, described recombinant protein A aggressiveness has the aminoacid sequence as shown in SEQIDNo.:5.
The present invention provides a kind of polynucleotide in second aspect, the described recombinant protein A aggressiveness of described polynucleotide encoding first aspect present invention item.
When known protein amino acid sequence, those skilled in the art can design the polynucleotide sequence of suitable encode such amino acid sequences as required, and are expressed.
In another preference, the sequence of described polynucleotide is as shown in SEQIDNO.:1.
A third aspect of the present invention provides a kind of carrier, and described carrier contains the polynucleotide described in second aspect present invention.
A fourth aspect of the present invention provides a kind of host cell, and described host cell contains in carrier described in third aspect present invention or its genome the polynucleotide be integrated with described in second aspect present invention.
A fifth aspect of the present invention provides a kind of method of producing recombinant protein A aggressiveness described in first aspect present invention, comprises step:
A () cultivates the host cell described in fourth aspect present invention, thus express the recombinant protein A aggressiveness described in first aspect present invention; With
(b) recombinant protein A aggressiveness from culture system described in isolated or purified.
A sixth aspect of the present invention provides a kind of affinity chromatography medium, and described affinity chromatography medium contains the recombinant protein A aggressiveness described in first aspect present invention.
In another preference, in described affinity chromatography medium, described recombinant protein A aggressiveness is fixed in or is incorporated into carrier.
In another preference, described recombinant protein A aggressiveness is incorporated into carrier by thioether bond coupling.
A seventh aspect of the present invention provides a kind of purification process, comprises step: for raw material to be purified, carries out affinity chromatography with the affinity chromatography medium described in sixth aspect present invention.
In another preference, described purifying is for antibody purification.
In another preference, described raw material to be purified is the liquid raw material (as nutrient solution, fermented liquid etc.) containing antibody to be purified.
Recombinant protein A of the present invention has higher alkali resistance, and the recombinant protein A described in employing can improve the stability of albumen in affinity chromatography process and maximum carrying capacity.
Should be understood that within the scope of the present invention, above-mentioned each technical characteristic of the present invention and can combining mutually between specifically described each technical characteristic in below (eg embodiment), thus form new or preferred technical scheme.As space is limited, tiredly no longer one by one to state at this.
Accompanying drawing explanation
Fig. 1 is the collection of illustrative plates of expression plasmid pET30a-4Z '.
Fig. 2 is for the e. coli bl21 (DE3) containing plasmid pET30a-4Z ' is through the SDS-PAGE electrophorogram of IPTG abduction delivering recombinant protein A aggressiveness, and wherein each swimming lane is respectively: 1: before induction, and 2: 2hr after induction, 3: 3hr after induction.
Fig. 3 is the elution chromatography figure that SP chromatography media catches recombinant protein A aggressiveness.
Fig. 4 is the alkali resistance test result of recombinant protein A aggressiveness of the present invention and restructuring wild-type protein A (rPA).Wherein, figure (a) be recombinant protein A aggressiveness of the present invention in the basic conditions 37 DEG C hatch the SDS-PAGE electrophorogram after different time, wherein the incubation time of each Lane Sample is respectively: 1:0hr, 2:4hr, 3:8hr, 4:16hr, 5:24hr; Figure (b) be restructuring wild-type protein A in the basic conditions 37 DEG C hatch the SDS-PAGE electrophorogram after different time, wherein the incubation time of each Lane Sample is respectively: 1:0hr, 2:4hr, 3:8hr, 4:16hr.
Embodiment
The present inventor is through extensive and deep research, be surprised to find that the aminoacid sequence by transformation wild-type S. aureus mycoprotein A (SpA), amino acid mutation by specific position is specific amino acid, can improve its alkali resistance significantly.In addition, compared to wild-type SpA, SpA recombinant protein of the present invention (recombinant protein A aggressiveness) in the basic conditions stability is better, and in affinity chromatography process, its protein load is higher, thus optimizes protein purification procedures.Complete the present invention on this basis.
Term " albumen of tool antibody binding capacity " can refer to that this albumen can be incorporated into the Fc fragment of immunoglobulin (Ig).But its albumen do not got rid of in conjunction with Fc fragment also can in conjunction with other region, such as, the Fab region of immunoglobulin (Ig).
In the present invention, suddenling change is defined by the numbering of switch, before be wild-type or nonmutationed amino acid, after be the amino acid of sudden change.Such as, be that aspartic acid is expressed as " Asn3Asp " at the asparagine mutation at position 3 place.
The albumen that term " Z fragment " obtains after referring to and changing the 29th amino acids (glycine) of wild-type SpA (E, D, A, B, C) B fragment into L-Ala, its aminoacid sequence is as shown in SEQIDNO.2.
Recombinant protein A aggressiveness
The invention provides a kind of recombinant protein A aggressiveness, described recombinant protein A aggressiveness is the polymer that the series connection of the Z fragment of two or more staphylococcus aureus protein A is formed, and at least one in each Z fragment is the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
In another preference, each in described Z fragment is the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
In another preference, described recombinant protein A aggressiveness is 2-20 aggressiveness, preferably 4-10 aggressiveness.
In another preference, described recombinant protein A aggressiveness is 4 aggressiveness, 6 aggressiveness, 8 aggressiveness, 10 aggressiveness.
In another preference, two Z fragments adjacent in described recombinant protein A aggressiveness are directly connected, or are connected by connection peptides.
In another preference, described recombinant protein A aggressiveness is made up of Z fragment and connection peptides; Described connection peptides is present between Z fragment, and described Z fragment is all the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
In another preference, described recombinant protein A aggressiveness has the structure shown in formula (I):
Pa-(Za-L)n-Za-Pb(I)
In formula,
Pa be nothing, Met, leader sequence, secretion peptide sequence, for the sequence label of purifying or its combination;
Za is the aminoacid sequence of the Z fragment of staphylococcus aureus protein A independently, and at least one Za is the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr;
L is nothing or connection peptides sequence independently;
N is the positive integer of >=1;
Pb is nothing, Cys, the sequence label of purifying or its combination.
In another preference, n is the positive integer of 1-50, preferably the positive integer of 2-20.
In another preference, all Za are the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
In another preference, the sequence of described Za comprises the sequence as shown in SEQIDNo.:3.
In another preference, the sequence of described Za has the sequence as shown in SEQIDNo.:3.
In another preference, described L is length 3-50 (preferably 3-20) amino acid whose connection peptides sequence.
In another preference, described L by not affecting or the Amino acid profile of the correct folding and conformation of not remarkably influenced albumen, such as glycine, L-Ala, Serine etc.
In another preference, described L is selected from lower group: QKDAVFP, ASTKGP.
In another preference, described recombinant protein A aggressiveness has structure shown in formula (II):
Met-Za-L-Za-L-Za-L-Za-Cys(II)
Wherein, Za and L definition is the same.
In another preference, described recombinant protein A aggressiveness is the albumen of tool antibody binding capacity.
In another preference, described antibody comprises IgG antibody (such as IgG1, IgG2, IgG3, IgG4), IgM antibody, IgA antibody.
In another preference, the C-terminal of described recombinant protein A aggressiveness is also containing amino-acid residue Cys.
In another preference, described recombinant protein A aggressiveness has the aminoacid sequence as shown in SEQIDNo.:5.
Recombinant protein A aggressiveness of the present invention, except referring to have the recombinant protein A activity of above-mentioned tool antibody binding capacity, there is aminoacid sequence as outside the albumen of SEQIDNO.5, also comprise have identical with described recombinant protein A or close function, the variant form of SEQIDNO.5 sequence.These variant forms include, but is not limited to: several (are generally 1-20, more preferably 1-10 or 1-8 or 1-5 or 1-3 or 1-2) aminoacid deletion, insertion and/or replacement, and add or disappearance one or several (being generally 1-20, more preferably 1-10 or 1-5) amino acid at C-terminal.Such as, in the art, when replacing with similar nature or similar amino acid, the function of protein can not usually be changed.Again such as, add at C-terminal the function that or several amino acid also can not change protein usually.In another preference, the C-terminal of recombinant protein A aggressiveness with the addition of an amino-acid residue Cys.
The variant form of albumen comprises: homologous sequence, conservative variant, allelic variant, natural mutation, under high or low stringency condition can with the albumen coded by the DNA of the DNA hybridization of the recombinant protein A of tool antibody binding capacity and the albumen utilizing the antiserum(antisera) of the recombinant protein A of described tool antibody binding capacity to obtain or albumen.
As used herein, unless otherwise indicated, described " recombinant protein A ", " SpA recombinant protein ", " recombinant protein A aggressiveness " is used interchangeably, and all refers to that wild-type SpA is through the improved recombinant protein of the present invention obtained of aminoacid sequence.
Recombinant protein A aggressiveness of the present invention can by chemosynthesis, or use recombinant technology to produce from protokaryon or eucaryon host.Host can be any cell (such as, bacterium, yeast, higher plant, insect and mammalian cell) of expressing recombinant protein well known by persons skilled in the art.In another preference, described host is e. coli bl21 (DE3).
In another preference, " Z fragment mutant " of the present invention is: by the 3rd of Z fragment the, 6,23 amino acids (l-asparagine) change aspartic acid into respectively, leucine and Threonine, obtain Z fragment mutant (as shown in SEQIDNO.3).
As used herein, term: " connection peptides " (linkerpeptide) refers to small peptide between two mutein fragments, that play ligation.The length of connection peptides is not particularly limited.The length of connection peptides is generally 5-50 amino acid.In another preference, connection peptides does not affect or not remarkably influenced mutein fragments forms correct folding and space conformation.In another preference, connection peptides sequence is approximately 5-50 amino acid.In another preference, connection peptides sequence is approximately 10-20 amino acid.In another preference, connection peptides comprises the sequence as shown in SEQIDNO.4.In another preference, connection peptides has the sequence as shown in SEQIDNO.4.
Present invention also offers the nucleotide sequence of described recombinant protein A aggressiveness of encoding.Correspondingly, present invention comprises and can be used for being expressed in the host of restructuring by prior art producing the protein DNA sequence of mutant.(based on the degeneracy of codon, the present invention includes all nucleotide sequences of described recombinant protein A aggressiveness of can encoding.) in another preference, described recombinant protein A aggressiveness comprises the nucleotide sequence as SEQIDNo.1.In another preference, described recombinant protein A aggressiveness has the nucleotide sequence as SEQIDNo.1.
The nucleotide sequence full length sequence of SpA recombinant protein of the present invention or the synthetic method of its fragment can be chemical synthesis or pcr amplification method or recombination method.In another preference, obtained by chemical synthesis during described nucleotide sequence.
The present invention relates to a kind of recombinant expression vector for expressing SpA recombinant protein, described expression vector comprises nucleotide sequence and the carrier sequence of described SpA recombinant protein of encoding.Term " recombinant expression vector " refers to bacterial plasmid well known to those skilled in the art, phage, yeast plasmid, vegetable cell is viral, mammalian cell is viral or other carriers.In a word, copy as long as can stablize in host, any plasmid and carrier may be used in the present invention.In another preference, described expression vector comprises one or more selected marker, to be provided for the phenotypic trait of host cell selecting to transform, Tetrahydrofolate dehydrogenase, neomycin resistance and the green fluorescent protein (GFP) cultivated as eukaryotic cell or for colibacillary kantlex or amicillin resistance.In another preference, described expression vector is selected from pGEX or pET serial carrier.In another preference, described expression vector is pET30a (+) carrier.
The present invention also provides a kind of host expressing SpA recombinant protein of the present invention, comprises recombinant expression vector of the present invention in described expressive host.Described host can be any express cell well known by persons skilled in the art, can be prokaryotic cell prokaryocyte, as bacterial cell; Or the eukaryotic cell such as low, as yeast cell; Or higher eucaryotic cells, as vegetable cell.In another preference, described host is e. coli bl21 (DE3).
Can carry out with routine techniques well known to those skilled in the art with recombinant DNA transformed host cell.When host be prokaryotic organism as intestinal bacteria time, the competent cell that can absorb DNA can be gathered in the crops at exponential growth after date, uses CaCl
2method process, step used is well-known in this area.Another kind method uses MgCl
2.If needed, transform and also can be undertaken by the method for electroporation.When host is eukaryote, can select following DNA transfection method: calcium phosphate precipitation, conventional mechanical methods is as microinjection, electroporation, liposome packaging etc.
The transformant obtained can be cultivated by ordinary method, expresses SpA recombinant protein of the present invention.According to host cell used, substratum used in cultivation can be selected from various conventional medium.Cultivate under the condition being suitable for host cell growth, when after host cell growth to suitable cell density, induce it to express target protein by suitable method (as temperature transition or chemical induction).
Therefore, the present invention also provides a kind of zymotechnique of expressing the host of SpA recombinant protein.Described technique comprises by a certain amount of kind of daughter bacteria after multistage seed culture, adds in fermention medium by a certain amount of inoculum size, after inductor induction certain hour, express recombinant protein.
In another preference, the inoculum size of described kind of daughter bacteria is 5%-15%.In another preference, the inoculum size of described kind daughter bacteria is 10%.In another preference, described inductor is lactose or IPTG.In another preference, described inductor is IPTG.In another preference, described induction time is 1-5hr.In another preference, described induction time is 2.5hr.
SpA recombinant protein of the present invention can be expressed in cell or on cytolemma or be secreted into extracellular.If need, can utilize its physics, chemistry with other characteristic, by various separation method abstraction and purification target protein.Above-mentioned Isolation and purification method is well known to those skilled in the art.The example of these methods includes, but are not limited to: conventional renaturation process, combination by albumen precipitation process (salting-out method), centrifugal, the broken bacterium of infiltration, super process, ultracentrifugation, sieve chromatography (gel-filtration), adsorption chromatography, ion exchange chromatography, high performance liquid chromatography (HPLC) and other various liquid chromatography (LC) technology and these methods.
Application
SpA recombinant protein of the present invention may be used for the affinity media preparing antibody purification.Therefore, the invention provides a kind of affinity chromatography medium that can be used for antibody purification, comprising affinity media, and be attached to the SpA recombinant protein of the present invention of described affinity media.
The present invention has no particular limits described affinity media, can be any affinity media being suitable for attachment protein A, include but not limited to: the carriers such as agarose (pearl), dextrane gel, Mierocrystalline cellulose, macroporous adsorbent resin.
The method that SpA recombinant protein of the present invention is attached on affinity media as aglucon in affinity chromatography process can be carrier combined techniques or physisorphtion or crosslinking or entrapping method.In another preference, the method that described albumen is connected with carrier is crosslinking.In another preference, described albumen and carrier are crosslinked together by thioether bond.
Major advantage of the present invention is:
SpA recombinant protein of the present invention good stability in the basic conditions, the binding ability of antagonist or antibody class albumen is strong, the affinity chromatography medium prepared by SpA recombinant protein of the present invention has higher dynamic carrying capacity, thus is conducive to simplifying protein purification procedures, improves purification efficiency.
Below in conjunction with specific embodiment, content of the present invention is made and further illustrates.Should be understood that these embodiments are only not used in for illustration of the present invention to limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usual conveniently condition is as people such as Sambrook, molecular cloning: lab guide (NewYork:ColdSpringHarborLaboratoryPress, 2001) said condition in, or according to the condition that manufacture business men is advised.
Embodiment 1: the preparation of recombinant protein A aggressiveness
A. the synthesis of recombinant protein A aggressiveness gene
Adopt the cDNA sequence (as shown in SEQIDNo.1) of full genome composite coding recombinant protein A aggressiveness, sequence 768bp (entrusting Shanghai Jierui Biology Engineering Co., Ltd to complete), comprise initiator codon ATG and terminator codon TAA, the sequence of synthesis connects into expression vector pET30a (+), obtain expression plasmid, called after pET30a-4Z ', as shown in Figure 1.
B. Sequence Identification and expression
Use CaCl
2method is by plasmid pET30a-4Z ' transformation of E. coli BL21 (DE3), in containing 50 μ g/mL kantlex (kanamycin, be called for short kana) the dull and stereotyped overnight growth of LB after, picking mono-clonal bacterium colony, in access LB liquid nutrient medium (50 μ g/mLkana) 37 DEG C, 200rpm shaking culture 8hr.Getting 2mL bacterium liquid is added in 100mLLB substratum (50 μ g/mLkana), 37 DEG C, when 220rpm shaking culture is 0.6-0.8 to OD600, adding inducer isopropylthio-β-D-thiogalactoside (IPTG) to final concentration is 0.3mM, be placed in shaking table 37 DEG C of abduction deliverings, after induction 2hr, 3hr, the centrifugal 10min of 10000rpm collects thalline respectively, abandons supernatant.Thalline adds damping fluid resuspended (50mMTris-HCl, pH7.5), and high-pressure homogenization is broken, and the centrifugal 30min of 10000rpm collects supernatant, 12% polyacrylamide gel (SDS-PAGE) electrophoresis detection.Get a part of bacterium liquid sample presentation simultaneously and carry out gene sequencing checking.
As shown in Figure 2, the recombinant protein A polymer molecular amount size of abduction delivering conforms to expection result.Gene sequencing result and theory completely the same.
(37 DEG C, 200rpm) are cultivated by bacterial strain correct for checking access 30mLLB substratum (50 μ g/mLkana).Stop cultivating when OD600 reaches about 0.7 ~ 0.8, be dispensed in 1mL cryopreservation tube by bacterium liquid, adding glycerine to final concentration is 15%.-70 DEG C of preservations, set up seed bank.
C. fermentative production
Ferment-seeded substratum is LB substratum, and fermentor tank batch culture based component is peptone, yeast powder, sodium-chlor, magnesium sulfate, fermentor tank feed-batch culture liquid is glucose, peptone, yeast powder, Sodium phosphate dibasic, SODIUM PHOSPHATE, MONOBASIC, ammonium sulfate, 10% defoamer, metal ion and VITAMIN.
Fermenting process is:
First order seed is cultivated: by 1% inoculum size, seed liquor being accessed liquid amount is in the triangular flask of 30mL/250mLLB substratum (50 μ g/mLkana), and being placed in shaking table (180rpm/min) 37 DEG C, to be cultured to OD600 be 0.2 ~ 0.3.
Secondary seed is cultivated: by 5% inoculum size, first order seed being accessed liquid amount is in the triangular flask of 200mL/500mLLB substratum (50 μ g/mLkana), and being placed in shaking table (180rpm/min) 37 DEG C, to be cultured to OD600 be 1 ~ 1.8.
To be added in fermentor tank by secondary seed by 10% inoculum size and start fermentative production, pH is set to 7.0, and temperature is 37 DEG C, and dissolved oxygen controls more than 20%.After inoculation, 2hr starts feed supplement, measures an OD600 and glucose content, add when OD600 reaches about 40 ~ 50 the IPTG that final concentration is 0.3mM every one hour, puts tank, collected by centrifugation thalline after induction 2.5hr, frozen.
D. purification protein sample
Thalline adds the resuspended (50mMTris-HCl of damping fluid, pH7.5), high-pressure homogenization is broken, the centrifugal 30min of 10000rpm, collect supernatant, with 0.22 μm of membrane filtration supernatant liquor, after low pH precipitates foreign protein, SP post loading, with 50mMTris-HCl, pH7.5 (NaCl containing 0.5M) gradient elution, obtains the recombinant protein A aggressiveness of purifying, is greater than 95% by the purity that HPLC detects.Color atlas as shown in Figure 3.
Embodiment 2: carrying capacity measures
The recombinant protein A aggressiveness prepared with embodiment 1 is for aglucon, and monodisperse porous multipolymer polystyrene-divinylbenzene (PS/DVB) microballoon is as carrier.Be in the PB damping fluid of 6 ~ 8 at pH, 25 DEG C of stirring reaction 8hr, albumen and microsphere supported by thioether bond be cross-linked, gained medium PB buffer solution several, remove non-associated proteins, vacuum-drying, obtain affinity chromatography medium (called after recombinant protein A polymer medium).
The cell conditioned medium sample (2.0g/L) of expressing human IgG is used to carry out carrying capacity mensuration to following affinity chromatography medium:
Recombinant protein A polymer medium;
MabCapture
tMmedium A (purchased from ABAppliedBiosystems company, article No. 4374730), medium as a comparison.
Sample-loading buffer is 10mMPB+0.2MNaCl, pH7.5.Elution buffer is 20mMSodiumCitrate+0.2MNaCl, pH3.7, and flow velocity is 115cm/h, carries out loading successively, wash-out.Elutriant is collected according to spectrogram.Adopt A280 method, measure the concentration of albumen in elutriant respectively, as shown in table 1, wherein, carrying capacity is pressed formula I and is calculated.
Carrying capacity=V1*C1/VcI
Wherein, V1 is effluent volume;
C1 is elutriant protein concentration;
Vc is column volume.
Table 1
Result is as shown in table 1, and the carrying capacity (19.0g/L) that result shows the recombinant protein A polymer medium that the present invention obtains significantly is better than MabCaptureA
tMcarrying capacity (7.9g/L).
Embodiment 5: recombinant protein A aggressiveness measures with restructuring wild-type protein A (rPA) alkali resistance
In recombinant protein A aggressiveness prepared by 10mL embodiment 1, (6mg/mL) adds 10mL1MNaOH solution mixing (in mixed solution, the concentration of NaOH is 0.5M), after hatch in 37 DEG C of thermostat containers, respectively at 4hr, 8hr, 16hr, 24hr sample, 12%SDS-PAGE electrophoretic analysis untreated (0hr) and the sample through base extraction, the sample applied sample amount of each time point is 5 μ g, and electrophoresis result is as shown in the figure (a) in Fig. 4.
Get 0.6mL restructuring wild-type protein A (the wild-type S. aureus mycoprotein A that E.coli is recombinant expressed, concentration is 1.2mg/mL) solution, add 5.4mL10mMPB, pH7.2, mixing, keep sample 1mL, isopyknic 1MNaOH solution mixing (in mixed solution, the concentration of NaOH is 0.5M) is added in remaining solution (5mL), hatch in 37 DEG C of thermostat containers, respectively at 4hr, 8hr, 16hr samples, 12%SDS-PAGE electrophoretic analysis untreated (0hr) and the sample through base extraction, the sample applied sample amount of each time point is 5 μ g, electrophoresis result is as shown in the figure (b) in Fig. 4.
Result shows, and recombinant protein A aggressiveness is hatched 16hr in the basic conditions and still had protein band, and after the wild-type protein A that recombinates hatches 4hr in the basic conditions, albumen is severely degrade.Illustrate that recombinant protein A aggressiveness of the present invention has stronger alkali resistance.
Embodiment 6: recombinant protein A polymer medium is to the mensuration of human IgG1's dynamic adsorption carrying capacity
Recombinant protein A polymer medium on-line cleaning test (CleaninginPlace prepared by embodiment 1, CIP), cleaning buffer solution is 0.5MNaOH, each circulating contact time is 10min, every 20 circulation mensuration dynamic adsorption carrying capacity (DynamicBindingCapacity, DBC).
DBC measuring method is as follows: sample is human IgG1, and flow velocity is 1mL/min, stops loading when penetrating to 10%.DBC is calculated according to formula II
10%.
DBC
10％＝(V
10％-V
0)*C/VcII
Wherein, V
10%it is 10% loading volume when penetrating;
V
0for residual sample volume in system pipeline;
C is sample concentration;
V
cfor column volume.
Result shows, and recombinant protein A polymer medium of the present invention is after 0.5MNaOH alkali lye 120 CIP, and DBC still retains more than 90%, shows that recombinant protein A polymer medium of the present invention has good alkali resistance.
The all documents mentioned in the present invention are quoted as a reference all in this application, are just quoted separately as a reference as each section of document.In addition should be understood that those skilled in the art can make various changes or modifications the present invention after having read above-mentioned teachings of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (11)
1. a recombinant protein A aggressiveness, it is characterized in that, described recombinant protein A aggressiveness is the polymer that the Z fragment series connection of two or more staphylococcus aureus protein A is formed, and at least one in each Z fragment is the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr.
2. recombinant protein A aggressiveness as claimed in claim 1, it is characterized in that, two Z fragments adjacent in described recombinant protein A aggressiveness are directly connected, or are connected by connection peptides.
3. recombinant protein A aggressiveness as claimed in claim 1, it is characterized in that, described recombinant protein A aggressiveness has the structure shown in formula (I):
Pa-(Za-L)n-Za-Pb(I)
In formula,
Pa be nothing, Met, leader sequence, secretion peptide sequence, for the sequence label of purifying or its combination;
Each Za is the aminoacid sequence of the Z fragment of staphylococcus aureus protein A independently, and at least one Za is the Z fragment mutant with following amino acid mutation: Asn3Asp, Asn6Leu and Asn23Thr;
L is nothing or connection peptides sequence independently;
N is the positive integer of >=1;
Pb is nothing, Cys, the sequence label of purifying or its combination.
4. recombinant protein A aggressiveness as claimed in claim 3, it is characterized in that, the sequence of described Za is as shown in SEQIDNo.:3.
5. recombinant protein A aggressiveness as claimed in claim 3, is characterized in that, described recombinant protein A aggressiveness has structure shown in formula (II):
Met-Za-L-Za-L-Za-L-Za-Cys(II)
Wherein, Za, L definition is the same.
6. polynucleotide, is characterized in that, recombinant protein A aggressiveness described in described any one of polynucleotide encoding claim 1-5.
7. a carrier, is characterized in that, described carrier contains polynucleotide according to claim 6.
8. a host cell, is characterized in that, described host cell contains in carrier according to claim 7 or its genome and is integrated with polynucleotide according to claim 6.
9. produce a method for recombinant protein A aggressiveness described in claim 1, it is characterized in that, comprise step:
A () cultivates host cell according to claim 8, thus express recombinant protein A aggressiveness according to claim 1; With
(b) recombinant protein A aggressiveness from culture system described in isolated or purified.
10. an affinity chromatography medium, is characterized in that, described affinity chromatography medium contains the recombinant protein A aggressiveness described in any one of claim 1-5.
11. 1 kinds of purification process, is characterized in that, comprise step: for raw material to be purified, carry out affinity chromatography with affinity chromatography medium according to claim 10.
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Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109311948A (en) * | 2016-05-11 | 2019-02-05 | 通用电气医疗集团生物工艺研发股份公司 | The method of cleaning and/or disinfection isolation medium |
| WO2020193485A1 (en) | 2019-03-26 | 2020-10-01 | Cytiva Sweden Ab | Method for sanitization of a chromatography column |
| WO2021109177A1 (en) * | 2019-12-06 | 2021-06-10 | 广州康盛生物科技股份有限公司 | Immunoglobulin binding protein, preparation method and application thereof |
| CN114409749A (en) * | 2022-03-15 | 2022-04-29 | 青岛科诺赛生物科技有限公司 | High-stability recombinant protein A with antibody binding force |
| WO2023046886A1 (en) | 2021-09-24 | 2023-03-30 | Cytiva Bioprocess R&D Ab | Fc binding polypeptides |
| WO2023174900A1 (en) | 2022-03-14 | 2023-09-21 | Cytiva Bioprocess R&D Ab | Vh3 binding polypeptides |
| RU2811436C1 (en) * | 2023-10-13 | 2024-01-11 | Общество С Ограниченной Ответственностью "Алтай-Энзим" (Ооо "Алтай-Энзим") | RECOMBINANT PLASMID pETZ, PROVIDING SYNTHESIS AND SECRETION OF RECOMBINANT PROTEIN A, AND RECOMBINANT PROTEIN A USED FOR IMMUNOCHEMICAL ASSAYS |
Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1642976A (en) * | 2002-03-25 | 2005-07-20 | 阿默森生物科学有限公司 | A mutated immunoglobulin-binding protein |
| CN103214563A (en) * | 2013-03-26 | 2013-07-24 | 江南大学 | Performance improved recombination staphylococcus aureus protein A affinity ligand and construction method thereof |
| WO2013147691A1 (en) * | 2012-03-28 | 2013-10-03 | Ge Healthcare Bio-Sciences Ab | Affinity chromatography matrix |
| CN103403020A (en) * | 2010-12-21 | 2013-11-20 | Jsr株式会社 | Novel alkali-resistant variants of protein a and their use in affinity chromatography |
-
2014
- 2014-09-16 CN CN201410471277.2A patent/CN105481954B/en active Active
Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1642976A (en) * | 2002-03-25 | 2005-07-20 | 阿默森生物科学有限公司 | A mutated immunoglobulin-binding protein |
| CN103403020A (en) * | 2010-12-21 | 2013-11-20 | Jsr株式会社 | Novel alkali-resistant variants of protein a and their use in affinity chromatography |
| WO2013147691A1 (en) * | 2012-03-28 | 2013-10-03 | Ge Healthcare Bio-Sciences Ab | Affinity chromatography matrix |
| CN103214563A (en) * | 2013-03-26 | 2013-07-24 | 江南大学 | Performance improved recombination staphylococcus aureus protein A affinity ligand and construction method thereof |
Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109311948A (en) * | 2016-05-11 | 2019-02-05 | 通用电气医疗集团生物工艺研发股份公司 | The method of cleaning and/or disinfection isolation medium |
| CN109311948B (en) * | 2016-05-11 | 2022-09-16 | 思拓凡生物工艺研发有限公司 | Method for cleaning and/or disinfecting a separation matrix |
| WO2020193485A1 (en) | 2019-03-26 | 2020-10-01 | Cytiva Sweden Ab | Method for sanitization of a chromatography column |
| WO2021109177A1 (en) * | 2019-12-06 | 2021-06-10 | 广州康盛生物科技股份有限公司 | Immunoglobulin binding protein, preparation method and application thereof |
| WO2023046886A1 (en) | 2021-09-24 | 2023-03-30 | Cytiva Bioprocess R&D Ab | Fc binding polypeptides |
| WO2023174900A1 (en) | 2022-03-14 | 2023-09-21 | Cytiva Bioprocess R&D Ab | Vh3 binding polypeptides |
| CN114409749A (en) * | 2022-03-15 | 2022-04-29 | 青岛科诺赛生物科技有限公司 | High-stability recombinant protein A with antibody binding force |
| RU2811436C1 (en) * | 2023-10-13 | 2024-01-11 | Общество С Ограниченной Ответственностью "Алтай-Энзим" (Ооо "Алтай-Энзим") | RECOMBINANT PLASMID pETZ, PROVIDING SYNTHESIS AND SECRETION OF RECOMBINANT PROTEIN A, AND RECOMBINANT PROTEIN A USED FOR IMMUNOCHEMICAL ASSAYS |
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