CN1974601A - New-type Fc fusion protein and its production process - Google Patents
New-type Fc fusion protein and its production process Download PDFInfo
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Abstract
The present invention provides one kind of Fc fusion protein and its production process, and relevant expression vector and engineering cell constructing, expressing and purifying process. The Fc fusion protein gene has the advantages of very stable secretion and expression in Pichia pastoris, high expression and purification yield, high stability, high expression level, and simple production process. The Fc fusion protein has high activity and long half life, and may be obtained in high efficiency, simple course and low cost.
Description
Technical field
The present invention relates to gene engineering technology field.More specifically, the invention provides a kind of New-type Fc fusion protein and expression method thereof, and the expression and the purifying process of the structure of relevant engineering cell, New-type Fc fusion protein.
Background
Many biomolecules with medicinal meaning, comprise cytokine, only produce moment and partial biological action in vivo, its loop cycle is relatively very short, for example, the transformation period of the Interferon, rabbit in the human serum just have only 2-8 hour (Roche labs.Referon A.Schering Intron A.Physicians ' desk Referece, 47th edition, pp.2006-2008,2194-2201).During the treatment disease, need long-term frequent drug administration, treatment cycle is long, not only brings misery to the patient, also can increase the toxic side effect of medicine simultaneously, and the medical expense height, has limited it and has further used and promote.In order to overcome these shortcomings, just need to modify this drug molecule, make it under the prerequisite that does not reduce pharmaceutical efficacy, increase its circulation time in blood.
At present, long-acting Study of cytokines approach mainly contains three kinds: PEG modifies, liposomal encapsulated and fusion protein F c technology.It is with macromole water-soluble polymers polyoxyethylene glycol (PEG) modified cytokines that PEG modifies, make and himself stablize and reduce their metabolic speed in vivo, its technology is simple, but productive rate is low, cost an arm and a leg, and, influenced albumen and reduced activity with combining of acceptor owing to the long chain polymer space steric effect; Liposomal encapsulated is with the lipid technology encapsulation of cells factor, and cytokine is that the form with slowly-releasing enters blood in the liposome, thereby prolongs its residence time in vivo; This technical sophistication, the encapsulation rate of liposome is low, when easy inactivation in the encapsulation process, liposome arrive interior target area of body or acceptor, the enrichment of high density can't occur, influences result of treatment.The Fc fusion protein technology is to utilize genetically engineered and protein engineering, amalgamation and expression cytokine-Fc fragment (IgG4).IgG4 is by Fc fragment and its receptors bind, and the ligand-receptor mixture was kept in vivo 20 days and not to be decomposed.Though this technology makes up complicated, purifying is simple, productive rate height, long half time.The Fc integration technology increases the transformation period of fusion rotein greatly, and application prospect is good, is the developing direction of depot drug product.
The medicine Enbrel of American I mmunex company and the development of American Home Products company uses the recombinant human P75 Tumor Necrosis Factor Receptors of expressing cho cell and human IgG1 Fc fusion rotein two concrete exactly.This medicine was in application listing in 1998, was used to alleviate the treatment of poisoning more than 4 years old to the reactivity patient with rheumatoid arthritis symptom of severe by the FDA approval in 1999.Be used to alleviate psoriasis arthropathica patient's symptom in 2002 by the FDA approval.
The invention provides a kind of New-type Fc fusion protein and production method thereof, adopt this New-type Fc fusion protein of CHO mammalian expression system secreting, expressing, by optimizing fermentation and purifying process, whole process stabilization, quick, easy obtains the Fc fusion rotein that activity is high, the transformation period prolongs.
Summary of the invention
Purpose of the present invention just provides a kind of New-type Fc fusion protein.
Another object of the present invention just provides the production method of this New-type Fc fusion protein, comprises the expression vector and the engineering cell that are used for this method.
In a first aspect of the present invention, just provided a kind of aminoacid sequence of New-type Fc fusion protein, it is characterized in that, its sequence is A-B-C or C-B-A, wherein A is an immunoglobulin fc region, from human normal immunoglobulin gamma-4 (IgG4) (seeing SEQ ID NO:1), have in blood of human body stable, efficient secretory expression and avoid such as advantages such as complementary reaction and ADCC, B is connection chain (Linker) (seeing SEQ ID NO:2), help to keep proteic native configurations and biological activity, C is a target protein, can but be not limited only to cytokine.
In a second aspect of the present invention, several expression vectors are provided, these expression vectors can be Yeast expression carrier or mammalian expression vector, it is characterized in that, described expression vector contains the encoding sequence of New-type Fc fusion protein mentioned above.
In another preference, described expression vector is the pPIC9k/ New-type Fc fusion protein.
In a third aspect of the present invention, several engineering cells are provided, can be yeast, mammalian cell and insect cell etc., it is characterized in that it is integrated with the described expression vector of claim 3.
In another preference, described engineering cell is a pichia spp.
In a fourth aspect of the present invention, a kind of method of producing recombined new Fc fusion rotein is provided, the method comprising the steps of:
C) under the expression condition that is fit to, cultivate host cell as claimed in claim 5, thereby give expression to New-type Fc fusion protein;
D) separation and purification New-type Fc fusion protein.
Description of drawings
Fig. 1 is that recombinant plasmid pPIC9K/Fc-IFNa-2b makes up synoptic diagram.
Embodiment
The inventor is extensive studies by going deep into, human normal immunoglobulin gamma-4 (IgG4) Fc district is linked to each other with target protein by connection chain, construct New-type Fc fusion protein, and be example with Interferon, rabbit a-2b, by protein expression research, obtained New-type Fc fusion protein Fc-IFNa-2b.Finished the present invention on this basis.
With immunoglobulin fc region, from human normal immunoglobulin gamma-4 (IgG4) Fc district (seeing SEQ IDNO:1), have in blood of human body stable, efficient secretory expression and avoid such as advantages such as complementary reaction and ADCC, by connection chain (Linker) (seeing SEQ ID NO:2), help to keep proteic native configurations and biological activity, be connected with target protein, obtain New-type Fc fusion protein.
According to the host cell of expressing, the encoding sequence of New-type Fc fusion protein is optimized, adopt conventional molecular biology method or complete sequence synthetic way to obtain required sequence then, be building up to corresponding expression vector then.Transform corresponding host cell.Obtain the high expression level engineering cell by resistance screening.
After obtaining engineering cell, just can be under the condition that is fit to the culturing engineering cell.In the present invention, the New-type Fc fusion protein expression condition of engineering cell expression is not particularly limited.Can adopt the culture condition of this area routine.
For the extensive New-type Fc fusion protein that obtains, need be optimized cultivation.The present invention has studied pilot scale and has optimized technology, and the expression level after the optimization can reach 500mg/L.
After expressing New-type Fc fusion protein, the New-type Fc fusion protein of expressing is carried out separation and purification.
Usually, cultivate earlier in modes such as centrifugal, filtrations and obtain culture supernatant.Supernatant can by saltout, method such as ultrafiltration carries out carrying out chromatography purification again behind the preliminary purification, also can directly carry out chromatography purification.
Be applicable to that chromatographic technique of the present invention comprises hydrophobic chromatography, ion exchange chromatography, gel permeation chromatography, affinity chromatography etc.
Through 2-3 step purifying, can obtain the pure product of New-type Fc fusion protein, the purifying yield is more than 30%, and purity is more than 95%, the about 150mg/L supernatant liquor of pure product yield.
New-type Fc fusion protein is again through determination of activity behind the purifying, and it is not less than the target protein activity that does not merge than living.
In an example of the present invention, be example with Interferon, rabbit a-2b, the structure of New-type Fc fusion protein and expression plasmid thereof is provided.
In another example of the present invention, obtained the bacterial strain of stability and high efficiency expression New-type Fc fusion protein by screening.
In another example of the present invention,, can obtain pure product 150mg/L through purifying process optimization.
Pilot scale research shows that product manufacture is stable, and is simple to operate, and the cycle is short, and cost is low, and every liter of fermented supernatant fluid can obtain the pure product 150mg of New-type Fc fusion protein.Be fit to industrialization production.
The invention has the advantages that:
(1) prolong half-life.The N end or the C end of target protein encoding sequence immunoglobulin fc region are connected, and in eukaryotic host cell, express, the target protein transformation period of generation is increased;
2) biological activity is not lost.Proteic native configurations and three-dimensional handiness thereof and biological activity are in close relations, and linker helps keeping native configurations and three-dimensional handiness thereof separately, thereby keeps its biological activity;
(2) by the crucial expression process condition of control, expression level is further improved.
(3) purifying process is easy, rate of recovery height.Owing to be secretory protein, therefore simplified purification procedures, the purifying rate of recovery is improved greatly, make the scale operation New-type Fc fusion protein become possibility.
Below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, the condition described in people such as Sambrook " molecular cloning: laboratory manual (New York:Cold Spring Harbor Laboratory Press; 1989) " for example, or the condition of advising according to manufacturer.
The structure of acquisition of embodiment 1 antigen-4 fusion protein gene and expression plasmid
Human normal immunoglobulin gamma-4 (IgG4) Fc district is linked to each other with target protein Interferon, rabbit a-2b by connection chain, construct New-type Fc fusion protein Fc-IFNa-2b, according to the yeast codon-bias, encoding sequence to New-type Fc fusion protein Fc-IFNa-2b is optimized, adopt complete sequence synthetic way to obtain required sequence then, pcr amplification IFN-a-2b, AAAGGA sequence before its 5 ' end has been introduced xhoI restriction enzyme site and α-factor leading peptide signal cracking site, 3 ' end is introduced the EcoRI restriction enzyme site.IFN-a-2b gene product and plasmid pPIC9 (available from Invitrogen company) that the pfu enzymatic amplification is obtained carry out double digestion (MBI, 2 with XhoI+EcoRI respectively
*TangoTM, 37 ℃), reclaim big fragment.Two fragments are connected with the T4 dna ligase, connect the product conversion and enter bacillus coli DH 5 alpha (available from Promega company), containing selected clone on the LB flat board of penbritin, preparing plasmid in a small amount, going out positive colony by double digestion/PCR evaluation and screening.
The pPIC9/Fc-IFN-a-2b plasmid of a large amount of amplification conclusive evidences is with BamHI+EcoRI double digestion (MBI, 1
*TangoTM, 37 ℃), reclaim small segment; Plasmid pPIC9K (available from Invitrogen company) handles with identical enzyme, reclaims big fragment.Two fragments are connected with the T4 dna ligase, connect the product conversion and enter bacillus coli DH 5 alpha, select positive colony on the LB flat board of penbritin and carry out plasmid enzyme restriction and identify containing, prepare plasmid in a small amount, go out positive colony by double digestion/PCR evaluation and screening.
Make up wiring diagram as shown in Figure 1.
Embodiment 2 high stables are expressed the screening of the bacterial strain of New-type Fc fusion protein
The recombinant plasmid pPIC9K/Fc-IFN-a-2b that builds is prepared in a large number, linearizing, electroporation transformed host cell P.pastoris GS115, coating contains the high anti-positive colony of YPDS plate screening of different concns microbiotic Zeocin, and carry out little megger and reach experiment, screening obtains the engineering yeast strain that high stable is expressed, and analyzes the engineering cell of the high stable expression that obtains, and the engineering yeast strain contains the nucleotide sequence of coding SEQ ID NO:3 aminoacid sequence.
Fermented liquid is carried out ultrafiltration and concentration, the buffer system of fermented liquid is replaced with phosphate solution (PB).Use the Millipore ultra-fine filter, the ultra-filtration membrane molecular weight that dams is 10KD, leaves and takes concentrated solution during ultrafiltration, adds PB, continues ultrafiltration; This program is flat identical with the PB damping fluid with pH until the conductivity water of sample repeatedly.
Chromatography 1: ion exchange chromatography
Chromatography media: SP-Sepharose FF
Method: the Fc-IFN-a-2b fermented liquid that contains after the ultrafiltration is crossed post.Wash chromatography column with phosphate buffered saline buffer behind the last sample, to OD
280<0.05.With 0-1M NaCl gradient with the target protein wash-out.
Chromatography 2: sieve chromatography
Chromatography media: Sephacryl S200
Damping fluid: phosphate buffered saline buffer
Behind the sample, cross chromatography column with PB solution constant speed on the Fc-IFN-a-2b sample peak that ion-exchange obtains, collection contains active protein peak.
This three steps purifying of sample process, i.e. ultrafiltration, after anion-exchange chromatography, the gel permeation chromatography, purity is increased to more than 95%, can get pure product 150mg/L.
All quote in this application as a reference at all documents that the present invention mentions, just quoted as a reference separately as each piece document.Should be understood that in addition 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 institute restricted portion equally.
Sequence table
<110〉Shanghai Xinshengyuan Medicine Research Co., Ltd.
<120〉a kind of New-type Fc fusion protein and production method thereof
<160>4
<210>1
<211>250
<212>PRT
<213〉homo sapiens
<223〉the segmental aminoacid sequence of the Fc of human normal immunoglobulin IgG4
<400>1
Met?Glu?Thr?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu?Trp?Val?Pro
1 5 10 15
Gly?Ser?Thr?Gly?Asp?Glu?Ser?Lys?Tyr?Gly?Pro?Pro?Cys?Pro?Ser?Cys
20 25 30
Pro?Ala?Pro?Glu?Phe?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro
35 40 45
Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys
50 55 60
Val?Val?Val?Asp?Val?Ser?Gln?Glu?Asp?Pro?Glu?Val?Gln?Phe?Asn?Trp
65 70 75 80
Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Lys?Thr?Lys?Pro?Arg?Glu
85 90 95
Glu?Gln?Phe?Asn?Ser?Thr?Tyr?Arg?Val?Val?Ser?Val?Leu?Thr?Val?Leu
100 105 110
His?Gln?Asp?Trp?Leu?Asn?Gly?Lys?Glu?Tyr?Lys?Cys?Lys?Val?Ser?Asn
115 120 125
Lys?Gly?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly
130 135 140
Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Gln?Glu?Glu
145 150 155 160
Met?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr
165 170 175
Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn
180 185 190
Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe
195 200 205
Leu?Tyr?Ser?Arg?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Glu?Gly?Asn
210 215 220
Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr
225 230 235 240
Gln?Lys?Ser?Leu?Ser?Leu?Ser?Leu?Gly?Lys
245 250
<210>2
<211>16
<212>PRT
<213〉artificial sequence
<223〉connection peptides
<400>2
Gly?Gly?Ser?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser
1 5 10 15
<210>3
<211>431
<212>PRT
<213〉artificial sequence
<223〉Fc-IFN-a-2b fusion rotein aminoacid sequence
<400>3
Met?Glu?Thr?Asp?Thr?Leu?Leu?Leu?Trp?Val?Leu?Leu?Leu?Trp?Val?Pro
1 5 10 15
Gly?Ser?Thr?Gly?Asp?Glu?Ser?Lys?Tyr?Gly?Pro?Pro?Cys?Pro?Ser?Cys
20 25 30
Pro?Ala?Pro?Glu?Phe?Leu?Gly?Gly?Pro?Ser?Val?Phe?Leu?Phe?Pro?Pro
35 40 45
Lys?Pro?Lys?Asp?Thr?Leu?Met?Ile?Ser?Arg?Thr?Pro?Glu?Val?Thr?Cys
50 55 60
Val?Val?Vsl?Asp?Val?Ser?Gln?Glu?Asp?Pro?Glu?Val?Gln?Phe?Asn?Trp
65 70 75 80
Tyr?Val?Asp?Gly?Val?Glu?Val?His?Asn?Ala?Als?Thr?Ths?Pro?Arg?Glu
85 90 95
Glu?Gln?Phe?Asn?Ser?Thr?Thr?Tvr?Arg?Val?Ser?sel?Leu?Thr?Val?Leu
100 105 110
His?Gln?Asp?Trp?Leu?Asn?Gly?Gls?Glu?Tvr?Lvs?Cys?Lvs?Val?Ser?Asn
115 120 125
Lys?Gly?Leu?Pro?Ser?Ser?Ile?Glu?Lys?Thr?Ile?Ser?Lys?Ala?Lys?Gly
130 135 140
Gln?Pro?Arg?Glu?Pro?Gln?Val?Tyr?Thr?Leu?Pro?Pro?Ser?Gln?Glu?Glu
145 150 155 160
Met?Thr?Lys?Asn?Gln?Val?Ser?Leu?Thr?Cys?Leu?Val?Lys?Gly?Phe?Tyr
165 170 175
Pro?Ser?Asp?Ile?Ala?Val?Glu?Trp?Glu?Ser?Asn?Gly?Gln?Pro?Glu?Asn
180 185 190
Asn?Tyr?Lys?Thr?Thr?Pro?Pro?Val?Leu?Asp?Ser?Asp?Gly?Ser?Phe?Phe
195 200 205
Leu?Tyr?Ser?Arg?Leu?Thr?Val?Asp?Lys?Ser?Arg?Trp?Gln?Glu?Gly?Asn
210 215 220
Val?Phe?Ser?Cys?Ser?Val?Met?His?Glu?Ala?Leu?His?Asn?His?Tyr?Thr
225 230 235 240
Gln?Lys?Ser?Leu?Ser?Leu?Ser?Leu?Gly?Lys?Gly?Gly?Ser?Gly?Gly?Ser
245 250 255
Gly?Gly?Gly?Gly?Ser?Gly?Gly?Gly?Gly?Ser?Cys?Asp?Leu?Pro?Gln?Thr
260 265 270
His?Ser?Leu?Gly?Ser?Arg?Arg?Thr?Leu?Met?Leu?Leu?Ala?Gln?Met?Arg
275 280 285
Arg?Ile?Ser?Leu?Phe?Ser?Cys?Leu?Les?Asp?Arg?His?Asp?Phe?Gly?Phe
290 295 300
Pro?Gln?Glu?Glu?Phe?Gly?Asn?Gln?Phe?Gln?Lys?Ala?Glu?Thr?Ile?Pro
305 310 315 320
Val?Leu?His?Glu?Met?Ile?Gln?Gln?Ile?Phe?Asn?Leu?Phe?Ser?Thr?Lys
325 330 335
Asp?Ser?Ser?Ala?Ala?Trp?Asp?Glu?Thr?Leu?Leu?Asp?Lys?Phe?Tyr?Thr
340 345 350
Glu?Leu?Tyr?Gln?Gln?Leu?Asn?Asp?Leu?Glu?Ala?Cys?Val?Ile?Gln?Gly
355 360 365
Val?Gly?Val?Thr?Glu?Thr?Pro?Leu?Met?Lys?Glu?Asp?Ser?Ile?Leu?Ala
370 375 380
Va1?Arg?Lys?Tyr?Phe?Gln?Arg?Ile?Thr?Leu?TTr?Leu?lys?Glu?Lys?Lys
385 390 395 400
Tyt?Ser?Pro?Cys?Ala?Trp?Glu?Val?Val?Arg?Ala?Glu?Ile?Met?Arg?Ser
405 410 415
Phe?Ser?Leu?Ser?Thr?Asn?Leu?Gln?Glu?Ser?Leu?Arg?Ser?Lys?Glu
420 425 430?431
Claims (7)
1. a New-type Fc fusion protein aminoacid sequence is characterized in that, its aminoacid sequence is A-B-C or C-B-A, wherein,
A is an immunoglobulin (Ig), from human normal immunoglobulin gamma-4 (IgG4) Fc fragment;
B is connection chain (Linker), and sequence is GGSGGSGGGGSGGGGS;
C is the purpose expressing protein, can but be not limited only to cytokine.
2. an expression vector is characterized in that, it contains the nucleotide sequence of the described aminoacid sequence of coding claim 1.
3. expression vector as claimed in claim 2 is characterized in that, it is a primary yeast or carrier for expression of eukaryon.
4. an engineering cell is characterized in that, it is integrated with the described expression vector of claim 3.
5. engineering cell as claimed in claim 4 is characterized in that, it is yeast or eukaryotic cell.
6. the production method of a New-type Fc fusion protein is characterized in that, the method comprising the steps of:
A) under the expression condition that is fit to, cultivate engineering cell as claimed in claim 5, thereby give expression to New-type Fc fusion protein;
B) separation and purification goes out the New-type Fc fusion protein of expression.
7. method as claimed in claim 6 is characterized in that, the expression condition shown in the step (b) is:
(1). fermentation or nutrient solution obtain to contain the supernatant liquor of target protein by simple centrifugal or ultrafiltration;
(2). by easy steps such as simple ion exchange chromatography, sieve chromatographies, can obtain purity at the pure product more than 95%.
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US11161889B2 (en) | 2018-04-09 | 2021-11-02 | Amgen Inc. | Growth differentiation factor 15 fusion proteins |
CN112646044A (en) * | 2020-12-25 | 2021-04-13 | 山东晶辉生物技术有限公司 | TFF2-Fc fusion protein and high-efficiency expression production method thereof |
CN112646044B (en) * | 2020-12-25 | 2022-12-27 | 山东睿鹰制药集团有限公司 | TFF2-Fc fusion protein and high-efficiency expression production method thereof |
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