CN106117324A - Recombinant protein A and encoding gene thereof and application - Google Patents

Recombinant protein A and encoding gene thereof and application Download PDF

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Publication number
CN106117324A
CN106117324A CN201610613358.0A CN201610613358A CN106117324A CN 106117324 A CN106117324 A CN 106117324A CN 201610613358 A CN201610613358 A CN 201610613358A CN 106117324 A CN106117324 A CN 106117324A
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CN
China
Prior art keywords
recombinant protein
protein
encoding gene
antibody
seq
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CN201610613358.0A
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Chinese (zh)
Inventor
韩振伟
宋炜
卢晅
张雨成
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Hubei Ai Sheng Biological Technology Co Ltd
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Hubei Ai Sheng Biological Technology Co Ltd
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Priority to CN201610613358.0A priority Critical patent/CN106117324A/en
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/195—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria
    • C07K14/305—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F)
    • C07K14/31—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from bacteria from Micrococcaceae (F) from Staphylococcus (G)
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
    • B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
    • B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
    • B01J20/28—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties
    • B01J20/28014—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof characterised by their form or physical properties characterised by their form
    • B01J20/28016—Particle form
    • B01J20/28021—Hollow particles, e.g. hollow spheres, microspheres or cenospheres

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  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Analytical Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Biochemistry (AREA)
  • Biophysics (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Medicinal Chemistry (AREA)
  • Molecular Biology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Peptides Or Proteins (AREA)

Abstract

The invention discloses a kind of recombinant protein A and encoding gene thereof and application, by the encoding gene of native protein A is transformed, the recombinant protein A expression making coding is significantly increased, and the recombiant protein after modification has certain toleration to the alkalescence extreme condition such as condition and protease.The adsorbent using this recombiant protein to prepare improves the absorption property of antagonist, and the Static Adsorptive capacity of human IgG is reached 50～60mg/g, improves the toleration to alkalescence condition simultaneously, and under the conditions of 0.1～0.5mol/L, tolerance time extends.

Description

Recombinant protein A and encoding gene thereof and application
Technical field
The present invention relates to a kind of recombinant protein A and encoding gene thereof and application, belong to biological technical field.
Background technology
Antibody class medicine includes the protein medicaments containing antibody gene segments, with target antigen can specific combination, There is the advantage such as the highest disease therapeuticing effect and the highest safety simultaneously, at neoplastic diseases, cardiovascular disease, especially It is that the therapeutic process of autoimmune disease progressively highlights important function, be that in present biological species medicine, rate of increase is the highest one Class medicine, plays an important role in field of biological pharmacy.
Adding up according to metaplasia thing database data, within 2006, global monoclonal antibody scale is only 20,000,000,000 dollars, and 2015 Year has reached 98,000,000,000 dollars, and in the preparation process of monoclonal antibody drug, the acquisition phase of antagonist is in the purifying process of antibody Play pivotal role.Now in the purifying process of antibody drug, more than 70% in application based on protein A derivative is all The short chain polypeptides class filler for antibody purification of row.Protein A is from A type staphylococcus aureus (Staphylococcus Aureus) a kind of cell wall protein being isolated, has the character can being combined with immunoglobulin, can be used for immunoassay With antibody and the purification of antibody class medicine.
Antibody drug purification filler based on protein A is of a great variety, but they are in antibody drug purge process, Easily there is series of problems in great majority: as carrying capacity during 1. antibody captures is relatively low, increase the cost of purification；2. antibody reclaims Rate is relatively low, and in elution process, the pH value of eluent is relatively low, causes partial antibody generation degeneration, reduces the response rate of antibody；3. Impurity is more, and much in CIP cleaning process, the time of NaOH incumbent firms is shorter, causes some impurity such as nucleic acid, endotoxin Remain more, virtually add the impurity in antibody, the most to a certain degree reduce the service life of filler simultaneously；4. aglucon takes off The rate that falls is high, and the expulsion rate of aglucon is heavily dependent on the coupling mode of aglucon, the amino acids distribution of aglucon, coupling mode Screening determines the degree that comes off of aglucon, and the unstable amino acid whose destruction of aglucon self also causes the increase that comes off simultaneously.5. filler Price is higher.These defects constrain the development of antibody drug to a certain extent.
Summary of the invention
When the invention aims to overcome existing protein A to be applied in antibody drug purification filler, antibody capture amount Defects the highest, that antibody changeableness, impurities left are many, aglucon expulsion rate is high, it is provided that a kind of recombinant protein A and encoding gene thereof and Application.
For achieving the above object, the recombinant protein A that the present invention provides, its aminoacid sequence such as SEQ ID NO:1, SEQ ID Shown in NO:2, SEQ ID NO:3, SEQ ID NO:4.
Present invention also offers the encoding gene of above-mentioned recombinant protein A, its nucleotide sequence is accordingly such as SEQ ID NO: 5, shown in SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8.
Present invention also offers the application in preparing antibody purification filler of above-mentioned recombinant protein A and encoding gene thereof.
Beneficial effects of the present invention:
1) through modifying improved aminoacid sequence, then after codon is optimized process, expression has and significantly carries High.
2) protein A after modifying has certain toleration to protease, reduces aglucon and comes off.
3) improve the adsorbent toleration to alkalescence condition, under the conditions of 0.1～0.5mol/L, tolerance time extends,
4) improve the absorption property of antagonist, the Static Adsorptive capacity of human IgG reaches 50～60mg/g.
Accompanying drawing explanation
Fig. 1 is the sequential design figure of the recombinant protein A of the present invention.
Fig. 2 is to be contrasted block diagram by the antibody purity prepared by recombinant protein A adsorption microspheres of the present invention.
Detailed description of the invention
Below in conjunction with specific embodiments and the drawings, the present invention is described in further detail.Method therefor in following embodiment It is conventional method if no special instructions.
The optimization of embodiment 1 protein A gene
As it is shown in figure 1, amino acid structure based on native protein A is analyzed, native protein A amino acid structure can divide Being the amino acid region of 5 very high homology, respectively E, D, A, B, C, there are about 50 aminoacid in each region, and especially B ties Structure territory, homology is the highest, and a lot of reports think that B structure territory is the key area of antibodies, and a lot of modification transformations are to tie based on B Structure territory, but our modification transformation, first, diamino acid helical structure based on B structure territory, add below simultaneously 8 sweet Amino acid sequence, is connected to the 3rd helix domain as linkage section, reduces by three spirals the most sterically hindered, improves absorption Performance.3rd helical structure uses C-structure territory and modifies it.
In the aminoacid sequence of protein A, each domain is three α spiral compositions, containing right in each domain These aminoacid are carried out modifying transformation and can improve alkaline resistance properties by the aminoacid of alkalescence condition responsive.Each domain contains Having about 8 agedoites, agedoite is the aminoacid the brightest to alkalescence condition, in order to strengthen its alkaline resistance properties, by No. 29 positions The glycine G29 amino acid mutation of point is tryptophan (Thr), maintains the stability of the structure of No. 28 site agedoites, with Time enhance alkaline resistance properties.Other agedoites, selection part carries out suddenly change N3A, N7D, N23T, to a certain degree can improve resistance to Alkalescence energy.
Aminoacid after modifying above carries out 3,4,5,6 repetitive sequence series connection, connects and at end with the form of series connection End adds cysteine, does point-to-point orientation coupling, improves coupling amount.
The aminoacid sequence designed, translates into nucleotide sequence and entrusts gene chemical synthesis company to carry out gene chemical synthesis, point Safety pin is to 3, and 4,5,6 repetitive sequences obtain SEQ ID NO:5～8.
The expression of embodiment 2 recombinant protein A
By the gene of above-mentioned synthesis carrier construction respectively, cultivate and the sequence pair that obtains of expression and purification answers SEQ ID NO:1 ～recombinant protein A 1 solution of 4, recombinant protein A 2 solution, recombinant protein A 3 solution, recombinant protein A 4 solution.
Employing expression vector is PET23a, single recombinant plasmid transformed to escherichia coli RosettaBlue (DE3) pLysS In carry out fermentation culture by LB fluid medium and carry out abduction delivering, after fermentation, collect thalline and use the side of thermal cracking Formula is destroyed cell wall and is discharged by expression product and centrifugation.
Being purified through IgG affinity media by separation liquid, separation liquid loading is through affinity media, with the phosphoric acid of 10mM Salt buffer cleansing medium, and collect destination protein with the buffer solution of pH3.8, and carry out being adjusted to neutral environment, preservation is treated With.The expression of above-mentioned carrier construction, cultivation and protein A and purification all use routine techniques, are well known to those skilled in the art, The most only sketch process.
Embodiment 3 recombinant protein A prepares antibody drug purification filler
Utilize embodiment 2 to express the recombinant protein A 1～A4 obtained and prepare recombinant protein A adsorption microspheres 1～4 respectively, preparation Process is as follows:
1) microsphere activation
Bio-sep-6FF, through purified water washing storage solution, drains in sand core funnel, weighs 10g microsphere, purified water 20ml, sodium hydroxide 0.2g, epoxychloropropane 10ml, react, 30 DEG C, 120rpm, constant temperature oscillator in 100ml conical flask Middle reaction 5h, completes epoxychloropropane and modifies activation.
2) coupling
In 100ml conical flask, add 80ml recombinant protein A solution (10mg/ml), add the microsphere 6FF after activation 10g, then it is separately added into Na2CO310mg, KCL 200mg, (NH4)2SO4500mg, NaOH 400mg, in constant temperature oscillator, 37 DEG C, 180rpm, 8h, it is then passed through chromatographic column and collects microsphere, the protein content in detection eluent, fill with PBS buffer solution Distinguish and wash gel, calculate coupling amount.
3) close
After coupling again by microsphere 10g and 20ml, 1mol/L ethanolamine solutions at 37 DEG C, 180rpm, react 2h, it is right to complete The closing of the epoxide group of residual, reduces impact.Completing adsorption microspheres preparation flow, adsorption microspheres coupling amount is about 10mg/g.
The IgG absorption property detection test of test example 1 recombinant protein A adsorption microspheres 1～4
This testing inspection step is as follows:
1) take 2g recombinant protein A adsorption microspheres to be encased in 10/20cm chromatographic column.
Level pad: 10mmol/L phosphate buffer+0.15M NaCL, pH7.0
Elution buffer: 0.1mol/L glycine buffer+0.15M NaCL, PH3.8
2) first the level pad of Ph 7.0 is rushed system to baseline steadily with the flow velocity of 2ml/min, then with 10ml/min's Flow velocity injects 20ml normal mouse ascitic fluid, cleans to baseline stability with the level pad of PH 7.0 after loading again.
3) carry out eluting with the elution buffer of PH 3.8 with the flow velocity of 5ml/min, collect eluent, drip in eluent Adding the Tris solution of pH8.5 1mo/L, regulation pH value is to neutral.
4) then cleaning filler with 0.5mol/L NaOH with the flow velocity of 2ml/min, scavenging period can extend to 30 points Clock, then clean with balance liquid, until baseline is steady.
5) finally use 20% ethanol purge, preserve wait and use next time.
In order to check the alkaline resistance properties of filler, according to above step, reuse, by IgG content in inspection eluent, Calculating absorption property, infer the alkaline resistance properties of filler, the testing result of recombinant protein A adsorption microspheres 1～4 see table.
Table 1. recombinant protein A adsorption microspheres 1～4 absorption property comparison sheet
The antibody purity detection of test example 2. recombinant protein A adsorption microspheres absorption
By antibody in each eluent in test example 1 is purified, recycle high pressure liquid chromatography SEC-HPLC pair It carries out purity detecting.(antibody-solutions is labeled as result display recombinant protein A adsorption microspheres by corresponding each adsorption microspheres numbering Antibody A 1～A4) antibody purity prepared is all more than 95%, and as shown in accompanying drawing 2, abscissa is antibody numbering, and vertical coordinate is pure Number of degrees value.
The aglucon of test example 3 recombinant protein A adsorption microspheres 1～4 comes off detection
By antibody in each eluent in test example 1 is purified, and detect with reference to commercially available Protein A ELISA Recombinant protein A in each antibody-solutions after purification, to rPA detection method, is detected, meter by test kit respectively Calculate the aglucon amount of coming off, the results are shown in Table 2.
The aglucon amount of coming off comparison sheet in table 2. recombinant protein A adsorption microspheres 1～4 solution
Above-mentioned test shows, the recombinant protein A aglucon expulsion rate prepared by the present invention is low, and absorption property is good.

Claims (10)

1. a recombinant protein A, its aminoacid sequence is as shown in SEQ ID NO:1.
2. a recombinant protein A, its aminoacid sequence is as shown in SEQ ID NO:2.
3. a recombinant protein A, its aminoacid sequence is as shown in SEQ ID NO:3.
4. a recombinant protein A, its aminoacid sequence is as shown in SEQ ID NO:4.
5. the encoding gene of recombinant protein A described in claim 1, its nucleotide sequence is as shown in SEQ ID NO:5.
6. the encoding gene of recombinant protein A described in claim 1, its nucleotide sequence is as shown in SEQ ID NO:6.
7. the encoding gene of recombinant protein A described in claim 1, its nucleotide sequence is as shown in SEQ ID NO:7.
8. the encoding gene of recombinant protein A described in claim 1, its nucleotide sequence is as shown in SEQ ID NO:8.
9. recombinant protein A application in preparing antibody purification filler described in any one of Claims 1 to 4.
10. the encoding gene of recombinant protein A described in any one of claim 5～8 application in preparing antibody purification filler.
CN201610613358.0A 2016-07-29 2016-07-29 Recombinant protein A and encoding gene thereof and application Pending CN106117324A (en)

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113278052A (en) * 2021-05-11 2021-08-20 平湖优谱生物技术有限公司 Preparation method of recombinant Protein A and affinity chromatography medium

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102365361A (en) * 2009-03-24 2012-02-29 株式会社钟化 Protein having affinity for immunoglobulin, and immunoglobulin-binding affinity ligand
CN102558317A (en) * 2002-03-25 2012-07-11 通用电气健康护理生物科学股份公司 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
CN103228328A (en) * 2010-11-29 2013-07-31 通用电气健康护理生物科学股份公司 Affinity chromatography matrix

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102558317A (en) * 2002-03-25 2012-07-11 通用电气健康护理生物科学股份公司 A mutated immunoglobulin-binding protein
CN102365361A (en) * 2009-03-24 2012-02-29 株式会社钟化 Protein having affinity for immunoglobulin, and immunoglobulin-binding affinity ligand
CN103228328A (en) * 2010-11-29 2013-07-31 通用电气健康护理生物科学股份公司 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 (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN113278052A (en) * 2021-05-11 2021-08-20 平湖优谱生物技术有限公司 Preparation method of recombinant Protein A and affinity chromatography medium
CN113278052B (en) * 2021-05-11 2022-09-09 博格隆(浙江)生物技术有限公司 Preparation method of recombinant Protein A and affinity chromatography medium

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Application publication date: 20161116