CN106928317A - A kind of new affinity chromatography medium and application - Google Patents

Abstract

The invention discloses a kind of new affinity chromatography medium and application.The Recombinant Staphylococal Protein A that the affinity chromatography medium that the present invention is provided is coupled comprising solid phase carrier and with solid phase carrier, the amino acid sequence such as SEQ ID NO of the Recombinant Staphylococal Protein A：Shown in 1.The affinity chromatography medium that the present invention is provided has protein binding high specificity, and affinity is high, purification efficiency advantage high, can be used for isolating and purifying for protein.

Description

A kind of new affinity chromatography medium and application

Technical field

The invention belongs to biological technical field, relate in particular to a kind of affinity chromatography medium and answer With.

Background technology

Affinity chromatography is a kind of protein separation technology.Some molecules in biomolecule Ad hoc structure position can be mutually distinguishable and combine with other molecules, such as the identification knot of enzyme-to-substrate The identification of conjunction, acceptor and part is combined, antibody is combined with the identification of antigen, and this combination is both Special, it is again reversible, change condition can release this combination.Affinity chromatography is just It is according to such principle design.

SP (Staphylococcus aureus protein A, SPA, protein A, Albumin A) it is aureus cell wall GAP-associated protein GAP, it has and the mankind and other food in one's mouths The Fc sections of ability of specific binding of immunoglobulin molecules, can adsorb in newborn animal blood IgG and the immune complex containing IgG.Based on these characteristics, protein A affinity chromatography post into It is the affinity column of the wide variety of antibody purification of biological technical field, is generally preparing albumin A During affinity chromatography medium, used Epichlorohydrin activation agarose all with water as reaction medium, Solubility of the epoxychloropropane in water is about 6.58% or so, therefore the reaction is a complexity Oil (epoxychloropropane) Gu-water-(gel) three-phase reaction system, reaction rate is slower；Simultaneously Easily there are hydrolytic side reactions in the basic conditions, it is necessary to reaction in epoxidation process epoxy group Condition carries out optimization comprehensively and strict control and could obtain activation efficiency higher.Shi Qinghong etc. leads to Cross and DMSO is introduced in reaction medium effectively eliminate epoxychloropropane agarose was activated Boundary in journey, makes epoxy base density effectively improve 100 μm of ol/mL or so, but Reaction medium is water, and hydrolysis is still more serious【Shi Qinghong etc..Epichlorohydrin activation agar Sugared gel process reinforcing and performance evaluation [J] process engineering journals, 2007,7 (4):743-746.】.

At present, with biotechnology industry, the especially fast development of designative species, egg The market demand of white A increases sharply, and affinity, purification efficiency to albumin A affinity column Etc. requiring more and more higher.But when the affinity chromatography technology containing albumin A is used, there is parent , purification efficiency low problem low with power, therefore when the affinity chromatography technology is applied, it is necessary to one Improved affinity chromatography medium is planted, purification effect is improved.

The content of the invention

The present invention develops a kind of new affinity chromatography medium by numerous studies.Specifically, of the invention Provide：

1st, a kind of affinity chromatography medium, it is characterised in that the affinity chromatography medium is comprising such as SEQ ID Recombinant protein A shown in NO.1.

2nd, the affinity chromatography medium described in above-mentioned 1, it is characterised in that also comprising solid phase carrier, institute Solid phase carrier is stated to be coupled with recombinant protein A.

3rd, the affinity chromatography medium described in above-mentioned 2, it is characterised in that the solid phase carrier is agar Sugared gel, glucan, cellulose, silica gel or hydroxyl high molecular polymer.

4th, the affinity chromatography medium described in above-mentioned 3, it is characterised in that the solid phase carrier is agar Sugar or sephadex.

5th, a kind of method for preparing any described affinity chromatography mediums of above-mentioned 1-4, methods described bag Include following steps：

(1) synthesis A gene of recombined protein monomer, 5 ' ends are designed by the method for chemical synthesis Ala-Asp is mutated into Asn-Val, to form AclI restriction enzyme sites, each A gene of recombined protein It is connected with AclI restriction enzyme sites between monomer, first A gene of recombined protein monomer front end adds The NcoI restriction enzyme sites being connected with expression vector, 6 His and EK restriction enzyme sites, last Individual A gene of recombined protein monomer end adds and stops codon TAA and and expression vector PET32a connected BamH I restriction enzyme sites；

(2) the A gene of recombined protein monomer digestion of step (1) is followed by into carrier pET32a's Between corresponding restriction enzyme site, then with AclI endonuclease digestions, screening is anti-with ampicillin Property transformant, through plasmid extraction, digestion screening is obtained and contains 4 protein A gene monomers SP expression vector pET32a-P；

(3) the expression vector pET32a-P of step (2) is converted into Escherichia coli with CaCl2 methods BL21DE3, screening, digestion obtain the sub- BL21DE3/pET32a-P of recombinant conversion；

(4) the bacterial strain BL21DE3/pET32a-P of step (3) is carried out into cultivation and fermentation, makes its efficient table Up to recombinant protein A, thalline is regathered, separated, purifying obtains recombinant protein A product；

(5) entered in media as well with epoxychloropropane, NaOH and agarose or sephadex Row reaction, the recombinant protein A that product is obtained with step (4) reacts at a temperature of 5-25 DEG C 15-20 hours, cleaning was drained again after having reacted, and obtains recombinant protein A gel.

6th, the preparation method described in above-mentioned 5, it is characterised in that step (5) Ago-Gel is 5% cross-linked agarose gel.

7th, the preparation method as described in above-mentioned 6, it is characterised in that in the step (5), epoxy chlorine Propane, NaOH and Ago-Gel are reacted in DMSO media.

8th, any described affinity chromatography mediums of above-mentioned 1-4 are applied in protein separation.

9th, the application described in above-mentioned 8, it is characterised in that the protein is antibody.

10th, the application described in above-mentioned 9, it is characterised in that the antibody is anti-egfr antibodies.

Affinity chromatography medium of the invention, its include solid phase carrier and with solid phase carrier be coupled Recombinant Staphylococal Protein A, wherein Recombinant Staphylococal Protein A nucleotide sequence is by 757 Individual nucleotides is constituted.Recombinant Staphylococal Protein A gene order of the present invention such as SEQ ID No.2 Shown, amino acid sequence is as shown in SEQ ID No.1.Test result indicate that, parent of the invention There is protein binding high specificity with chromatography media, affinity is high, purification efficiency advantage high, Compared with commercially available SP- Ago-Gels FF, its Dynamic Adsorption carrying capacity is significantly improved.

Specific embodiment

The present invention is further illustrated with reference to embodiments, and following embodiments should not be construed as to this The limitation of invention.Embodiment does not include detailed descriptions of conventional methods, and such as those are used to build The method of carrier and plasmid, the gene of encoding proteins is inserted into the side of such carrier and plasmid Plasmid is introduced method the method for host cell and the cell fusion and monoclonal antibody of classics Method of screening and purifying etc..Such method is for person having ordinary skill in the art It is it is well known that and being all described in many publications.

The preparation of the Recombinant Staphylococal Protein A of embodiment 1

1st, Recombinant Staphylococal Protein A gene monomer is built

By the method for chemical synthesis, design synthesis SP one repetition of gene Fragment (repeated fragment sequence such as SEQ ID NO:Shown in 47-217 nucleotides shown in 2) Sequence monomers, it include length be 171bp repeated fragment, and front end add with expression The connected NcoI restriction enzyme sites of carrier, 6 His (be used for affinity chromatography, combined with nickel post, Reduce purification step), EK restriction enzyme sites (for His and DDDDK to be cut, are formed just True Protein A) and AclI restriction enzyme sites (AACGTT, for being connected into multiple repetition pieces Section).In addition, also include terminator codon TAA, and clone's BamH I restriction enzymes The common 9bp of enzyme joint.Labeled as " monomer 1 ".

In addition, the method for passing through chemical synthesis, design synthesis SP gene one Individual repeated fragment (repeated fragment sequence such as SEQ ID NO:2 47-217 nucleotides institute Show) sequence monomers, its sequence includes that length is 171bp repeated fragments, and ends A clI Restriction enzyme site.Labeled as " monomer 2 ".

2nd, the expression vector of Recombinant Staphylococal Protein A gene is built

The monomer 1 obtained with restricted type restriction endonuclease NcoI and BamH I digestions embodiment 1, Then it is connected with through the carrier pET32a of NcoI and BamH I digestions, converts Escherichia coli, Transformant of the screening with amicillin resistance, through plasmid extraction, Portugal is proved after digestion identification Grape pneumococcal proteins A gene monomers have been cloned into pET32a, so as to successfully build one Portugal of sweat The pET32a carriers of grape pneumococcal proteins A gene monomers.

3rd, the structure of the pET32a-P carriers containing SP gene monomer

The monomer 2 that above-mentioned steps 1 are obtained reclaims respective segments, so with AclI digestions Afterwards grape globulin A gene monomer is recombinated with constructed by above-mentioned steps 2 containing one PET32a carriers are connected, and convert Escherichia coli, conversion of the screening with amicillin resistance Son, through plasmid extraction, digestion screening is obtained and contains 4 grape balls of protein A gene monomer The expression vector of mycoprotein A, labeled as " pET32a-P ", after testing, pET32a-P expression Carrier is comprising such as SEQ ID No：Gene order shown in 2.

4th, the coli strain of construction expression Recombinant Staphylococal Protein A

Use CaCl₂The pET32a-P conversion BL21DE3 that method is obtained above-mentioned steps 3, Transformant is screened on LB flat boards containing ampicillin, is obtained through plasmids detection and restriction analysis Recombinant conversion that must be containing pET32a-P is sub, is masked as " BL21DE3/pET32a-P ".

5th, the production of Recombinant Staphylococal Protein A and purifying

(1) cultivation and fermentation of strain

Picking colibacillus engineering BL21DE3/pET32a-P, is inoculated in LB culture mediums, 1~2%V/V of inoculum concentration, in 35 DEG C of overnight incubations, next day is aseptically by above-mentioned culture Good seed culture medium presses l:8-1:4 are inoculated on fermentation medium, 35 DEG C of fermentations to O.D600 0.4~0.7 is reached, 48 DEG C of inductions are warming up to, is centrifuged after 3~5 hours and is received bacterium.

Take a small amount of cell and plus 2 × sample-loading buffer, carry out PAGE gel electrophoresis detection, Result display SP has induced soluble-expression.

(2) purifying of Recombinant Staphylococal Protein A

With phosphate buffer, (0.2M, pH7.0-8.0 contain the thalline that step (1) is collected 0.1M NaCl) suspend, supernatant is collected in ultrasonication, 4 DEG C of centrifugations, obtains crude extract.

Crude extract is used into SDS-PAGE electrophoresis detections, most of staphylococcus is as a result shown Albumin A is slightly carried, and the amount for remaining in thalline is atomic：Crude extract is carried out into Sephacryl 5200 Molecular sieve purification, collects the restructuring protein staphylococcus that characteristic peak (the second eluting peak) is purifying A (is named as " Protein A 1 "), after testing, the amino acid sequences of Protein A 1 such as SEQ ID No：Shown in 1.

The ELISA method detection Recombinant Staphylococal Protein A of embodiment 2 and antibody binding activity

The Protein A 1 prepared using ELISA method detection above-described embodiment 1 are combined with antibody Activity, detailed process is as follows：

1) it is coated with：In 96 orifice plates per hole the sample 200u1 of coating Protein A 1,38 DEG C, 1h；

2) close：Closed with the 1% of 200u1 BSA per hole, 38 DEG C, 1h；

3) primary antibody is added：About 150ug human IgG antibodies (being purchased from Mlbio companies) are added per hole, 38 DEG C, 1h；

4) secondary antibody is added：About 200u1,1 are added per hole:1000 Horseradish Peroxidase Conjugates (being purchased from Mlbio companies), 38 DEG C, 1h；

5) develop the color.

Same method detects commercially available Protein A (purchased from an ancient biology) and antibody binding activity.

ELISA testing results：Protein A 1 of the invention are combined with human IgG antibody, per hole Coating 0.8-1.9ng SPs can obtain obvious detection signal, and commercially available Protein A just obtain obvious detection when needing to be coated with per hole 7.5-10.5ng SPs Signal.It can be seen that Protein A1 of the invention have stronger antibody binding activity.

The preparation of the Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium of embodiment 3

Ago-Gel affinity chromatography medium is prepared using the Protein A1 of embodiment 1, including Following steps：

1st, the activation of Ago-Gel：With epoxychloropropane, NaOH and agarose (5% Cross-linked agarose gel) in dimethyl sulfoxide (DMSO) (DMSO, purchased from toray group) medium In reacted, at a temperature of 45-60 DEG C react 2-3 hours, use distilled water after having reacted Drained after cleaning to neutrality, obtain the Ago-Gel of activation；

2nd, the chemical coupling of Recombinant Staphylococal Protein A and the Ago-Gel of activation：Will be upper The activated sepharose and the Protein A1 of the preparation of embodiment 1 for stating step 1 acquisition exist Reacted 15-20 hours at a temperature of 10-28 DEG C, cleaning is drained after having reacted, and obtains staphylococcus Protein A Sepharose beads affinity chromatography medium.

After testing, the Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium of preparation Characteristic is as shown in table 1：

Table 1, Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium characteristic

The preparation of the Recombinant Staphylococal Protein A sephadex affinity chromatography medium of embodiment 4

Sephadex affinity chromatography medium is prepared using the Protein A1 of embodiment 1, including Following steps：

1st, reacted in aqueous medium with epoxychloropropane, NaOH and sephadex, Reacted 2-3 hours at a temperature of 45-60 DEG C, cleaned to neutrality with distilled water after having reacted Drain, obtain the sephadex of activation；

Protein prepared by the activated dextran gel and embodiment 1 for the 2, obtaining above-mentioned steps 1 A1 is reacted 15-20 hours at a temperature of 10-28 DEG C, and cleaning is drained again after having reacted, and is obtained final product To SP sephadex affinity chromatography medium.

After testing, the Recombinant Staphylococal Protein A sephadex affinity chromatography medium of preparation Characteristic is as shown in table 2：

Table 2, Recombinant Staphylococal Protein A sephadex affinity chromatography medium characteristic

The affinity chromatography medium of embodiment 5 isolates and purifies antibody

It is affine using the Recombinant Staphylococal Protein A prepared in above-described embodiment 3, embodiment 4 Chromatography media isolates and purifies antibody (according to described in 201010148069.0 from antibody nutrient solution Method prepare monoclonal antibody against EGFR), detailed process is as follows：

1st, Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium isolates and purifies antibody

1), the affine layer of Recombinant Staphylococal Protein A Ago-Gel prepared by above-described embodiment 3 Analysis medium dress post, 1.6 × 20cm, bed volume is l0ml；

2), 5-10 bed body is balanced with buffer A (PBS solution of pH7.4, as follows) Product, flow velocity is l ml/min；

3) 2m1 monoclonal antibody against EGFR nutrient solution buffer As, are diluted to 20m1, 0.45 μm of membrane filtration, loading, flow velocity is l ml/min；

4) 5-10 bed volume, is washed again with buffer A, flow velocity is l ml/min；

5), with buffer B, (the 20mM citrate buffer solutions of pH4.0, its compound method is such as Under：Citric acid 2.1g is added water 950m1, and pH4.0 is adjusted to 5M NaOH, adds water to 1000 Ml) elute, flow velocity is l ml/min, collects eluting peak；

6) 10 bed volumes, are washed with stream of pure water, then 10 posts beds is washed with 20% ethanol stream Volume, flow velocity is 2ml/min, and pillar is placed in 4-8 DEG C of environment and preserves；

7) monoclonal antibody against EGFR and the reference substance that, will be isolated and purified are while carry out SDS-PAGE electrophoretic analysis.

(remarks：Pillar should be used to the buffer solution C (0.5M of pH3.0 after often using several times Acetate buffer solution, its compound method is as follows：0.5M acetum solids NaOH is adjusted to PH3.0) stream is washed 1 time, will to adsorb more firm albumen removal.)

SDS-PAGE electrophoresis results：Recombinant Staphylococal Protein A Ago-Gel of the invention Affinity chromatography medium once purifies the monoclonal antibody against EGFR purity about 97.2% of acquisition.

2nd, Recombinant Staphylococal Protein A sephadex affinity chromatography medium isolates and purifies antibody

Use the Recombinant Staphylococal Protein A sephadex parent prepared in above-described embodiment 4 Antibody is isolated and purified from antibody nutrient solution (according to 201010148069.0 institutes with chromatography media The method stated prepares monoclonal antibody against EGFR), detailed process is as follows：

1), the affine layer of Recombinant Staphylococal Protein A sephadex prepared by above-described embodiment 4 Analysis medium dress post, 1.6 × 20cm, bed volume is l0ml；

2), 5-10 bed body is balanced with buffer A (PBS solution of pH7.4, as follows) Product, flow velocity is l ml/min；

3) 2m1 monoclonal antibody against EGFR nutrient solution buffer As, are diluted to 20m1, 0.45 μm of membrane filtration, loading, flow velocity is l ml/min；

4) 5-10 bed volume, is washed again with buffer A, flow velocity is l ml/min；

5), with buffer B, (the 20mM citrate buffer solutions of pH4.0, its compound method is such as Under：Citric acid 2.1g is added water 950m1, and pH4.0 is adjusted to 5M NaOH, adds water to 1000 Ml) elute, flow velocity is l ml/min, collects eluting peak；

6) 10 bed volumes, are washed with stream of pure water, then 10 posts beds is washed with 20% ethanol stream Volume, flow velocity is 2ml/min, and pillar is placed in 4-8 DEG C of environment and preserves；

7) monoclonal antibody against EGFR and the reference substance that, will be isolated and purified are while carry out SDS-PAGE electrophoretic analysis.

(remarks：Pillar should be used to the buffer solution C (0.5M of pH3.0 after often using several times Acetate buffer solution, its compound method is as follows：0.5M acetum solids NaOH is adjusted to PH3.0) stream is washed 1 time, will to adsorb more firm albumen removal.)

SDS-PAGE electrophoresis results：Recombinant Staphylococal Protein A sephadex of the invention Affinity chromatography medium once purifies the monoclonal antibody against EGFR purity about 96.3% of acquisition.

3rd, commercially available SP- Ago-Gels FF isolates and purifies antibody

In addition, using identical method, (Bo Er is purchased from using commercially available SP- Ago-Gels FF Western scientific ＆ technical corporation) antibody is isolated and purified from nutrient solution (according to described in 201010148069.0 Method prepare monoclonal antibody against EGFR).SDS-PAGE electrophoresis results show, use MabSelectSure once purifies the monoclonal antibody against EGFR purity about 87% of acquisition.

Test result indicate that.Recombinant Staphylococal Protein A Ago-Gel of the invention is affine Chromatography media and the step of Recombinant Staphylococal Protein A Ago-Gel affinity chromatography medium one are with regard to energy Monoclonal antibody against EGFR of the purity more than 96% is obtained, its purification effect is substantially better than commercially available Product.

The detection of the Dynamic Adsorption carrying capacity of the affinity chromatography medium antagonist of embodiment 6

Affinity chromatography medium of the invention (is purchased from Bo Erxike with commercially available SP- Ago-Gels FF Skill company) antagonist Dynamic Adsorption carrying capacity compares (according to the side described in 201010148069.0 Method prepares monoclonal antibody against EGFR), step is as follows：

1st, 5-10 bed volume, flow velocity are balanced with buffer A (PBS solution of pH7.4) It is l ml/min；

2nd, 2mg/ml concentration monoclonal antibody against EGFR loading, flow velocity is l ml/min, extremely 10% stops when penetrating；

3rd, according to sample concentration, loading volume and column volume calculate 10% penetrate when each gel Dynamic Adsorption carrying capacity.

Experimental result：Recombinant Staphylococal Protein A Ago-Gel antagonist of the invention Dynamic Adsorption carrying capacity is for about 45.2mg/ml, Recombinant Staphylococal Protein A glucan of the invention The Dynamic Adsorption carrying capacity of gel antagonist is for about 42.6mg/ml, commercially available SP- Ago-Gels FF Dynamic Adsorption carrying capacity about 24mg/ml.

Test result indicate that, the Dynamic Adsorption carrying capacity of affinity chromatography medium antagonist of the invention is bright The aobvious Dynamic Adsorption carrying capacity higher than commercially available SP- Ago-Gels FF, with preferably purifying Effect.

Claims (10)

1. a kind of affinity chromatography medium, it is characterised in that the affinity chromatography medium is comprising such as SEQ ID Recombinant protein A shown in NO.1.

2. affinity chromatography medium according to claim 1, it is characterised in that also comprising solid phase Carrier, the solid phase carrier is coupled with recombinant protein A.

3. affinity chromatography medium according to claim 2, it is characterised in that the solid phase is carried Body is Ago-Gel, glucan, cellulose, silica gel or hydroxyl high molecular polymer.

4. affinity chromatography medium according to claim 3, it is characterised in that the solid phase is carried Body is agarose or sephadex.

5. a kind of method for preparing any described affinity chromatography mediums of claim 1-4, the side Method is comprised the following steps：

(1) synthesis A gene of recombined protein monomer, 5 ' ends are designed by the method for chemical synthesis Ala-Asp is mutated into Asn-Val, to form AclI restriction enzyme sites, each A gene of recombined protein list It is connected with AclI restriction enzyme sites between body, first A gene of recombined protein monomer front end adds and table Up to the NcoI restriction enzyme sites that carrier is connected, 6 His and EK restriction enzyme sites, last weight Histone A gene monomers end add stop codon TAA and with expression vector pET32a phases BamH I restriction enzyme sites even；

(2) the A gene of recombined protein monomer digestion of step (1) is followed by the phase into carrier pET32a Answer between restriction enzyme site, then with AclI endonuclease digestions, screening is with amicillin resistance Transformant, through plasmid extraction, digestion screening is obtained and contains 4 grapes of protein A gene monomer The expression vector pET32a-P of pneumococcal proteins A；

(3) the expression vector pET32a-P of step (2) is converted into Escherichia coli with CaCl2 methods BL21DE3, screening, digestion obtain the sub- BL21DE3/pET32a-P of recombinant conversion；

(4) the bacterial strain BL21DE3/pET32a-P of step (3) is carried out into cultivation and fermentation, makes its efficient Expression recombinant protein A, regathers thalline, and separated, purifying obtains recombinant protein A product；

(5) carried out in media as well with epoxychloropropane, NaOH and agarose or sephadex Reaction, the recombinant protein A that product is obtained with step (4) reacts at a temperature of 5-25 DEG C 15-20 hours, cleaning was drained again after having reacted, and obtains recombinant protein A gel.

6. preparation method according to claim 5, it is characterised in that step (5) agar Sugared gel is 5% cross-linked agarose gel.

7. preparation method according to claim 6, it is characterised in that in the step (5), Epoxychloropropane, NaOH and Ago-Gel are reacted in DMSO media.

8. according to any described affinity chromatography mediums of claim 1-4 in protein separation Application.

9. application according to claim 8, it is characterised in that the protein is antibody.

10. application according to claim 9, it is characterised in that the antibody is anti-EGFR Antibody.