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CN113061589A - Preparation method and application of cell strain for stably expressing biotin ligase Bir A - Google Patents

Preparation method and application of cell strain for stably expressing biotin ligase Bir A Download PDF

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CN113061589A
CN113061589A CN202110288528.3A CN202110288528A CN113061589A CN 113061589 A CN113061589 A CN 113061589A CN 202110288528 A CN202110288528 A CN 202110288528A CN 113061589 A CN113061589 A CN 113061589A
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bir
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recombinant protein
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biotinylation
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韩照中
潘红芽
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Linnuo Shanghai Pharmaceutical Technology Co ltd
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Abstract

The invention discloses a preparation method of a cell strain for stably expressing biotin ligase Bir A and application of the cell strain in site-specific biotinylation of recombinant protein. The invention provides a construction method of the cell strain and an application example thereof: inserting the Bir A gene into a eukaryotic expression vector; transfecting host cells to obtain a cell line for stably expressing BirA; constructing a recombinant protein expression vector with an Avi-protein purification combined label; the BirA cell line is transfected for producing in vivo site-directed biotinylated proteins. The invention has the unique characteristics that the expression of the BirA gene in eukaryotic mammal cells and the in-vivo site-specific biotinylation of recombinant protein are realized; HEK293F is selected as a host cell, so that fixed-point biotinylation in the cell is conveniently carried out while recombinant protein is produced, and the subsequent biological function detection efficiency is improved; provides a basic method and a technical approach for detecting and identifying in vivo biotinylated recombinant protein by using HEK293F-Bir A cell strain production.

Description

Preparation method and application of cell strain for stably expressing biotin ligase Bir A
Technical Field
The invention relates to a preparation method of a stable expression Bir A cell strain and application thereof in biotinylation in recombinant protein production.
Background
The Biotin-Avidin System (BAS) is a new type of amplification System for biological reactions developed in the late 70 s. The strong binding with high affinity between biotin and avidin and the multi-stage amplification effect make BAS immune labeling and related tracing analysis more sensitive. It has become a new technology widely used for qualitative and quantitative detection and positioning observation research of trace antigens and antibodies. Biotinylation by in vitro chemical methods is currently one of the most commonly used methods. The method is convenient and rapid, and has high biotinylation labeling efficiency. The disadvantage is that during the biotinylation of the protein reagent, the biotinylation sites are randomly labeled, and the steric hindrance caused by biotinylation of some sites can affect the activity of the protein. How to solve the defect is one of the problems to be solved in the research of the biological activity of the protein.
Biotin Ligase (Biotin Protein Ligase) is used to catalyze biotinylation reactions, which allows Biotin to be ligated to specific amino acid sites. Common types include Bir a in e. Bir A as biotin- - [ acetyl-CoA carboxylase ] ligase Bir A activates biotin in the presence of ATP to form a Bir A-biotin-5 '-adenylate (Bir A-bio-5' -AMP) complex, which is covalently bonded to a lysine site of the protein. The Avi-Tag protein is a short 15 amino acid peptide (GLNDIFEAQKIEWHE) with a single biotinylated lysine site, which is completely different from the known naturally biotinylated sequences and can be added to the N-and C-termini of the target protein. After expression of the fusion, it can be biotinylated by biotin ligase. Thus, with the aid of Avi-tag and biotin ligase, almost all proteins can be biotinylated efficiently and specifically at a unique Avi-tag site, either in vitro or in vivo, greatly reducing the steric hindrance effect due to biotinylation, to avoid affecting the biological functional activity of the protein.
Biotin ligase is derived from lower organisms such as bacteria, and is absent from higher mammalian cells. According to the invention, the expression of the Bir A gene in eukaryotic mammalian cells is realized, and the in-vivo fixed-point biotinylation is carried out in the protein production process, so that the efficiency of subsequent protein biological function detection can be greatly improved; meanwhile, the in vivo fixed point biotinylation is obviously superior to the in vitro chemical labeling method in keeping the biological function activity of the recombinant protein.
Disclosure of Invention
The invention provides a stable expression Bir A cell line with functional activity and a preparation method and application thereof.
In one aspect, the invention provides a eukaryotic cell line stably expressing BirA with functional activity
Biotin ligase is derived from lower organisms such as bacteria, and is absent from higher mammalian cells. Therefore, the invention inserts (1) Bir A gene into eukaryotic expression vector, introduces into HEK293F cell, and obtains cell line for stably expressing Bir A through pressure screening; (2) simultaneously constructing a protein expression vector with an Avi-protein purification label; (3) and (3) introducing the protein expression vector constructed in the step (2) into the Bir A cell line established in the step (1) for producing in-vivo site-directed biotinylated protein. The method comprises the following specific steps:
the Bir A gene is cloned to a eukaryotic expression vector, and the eukaryotic expression vector has the following characteristics:
the Bir A coding gene is under the control of a transcription regulatory element and can be expressed in mammalian cells;
the eukaryotic expression vector has all regulatory elements required for transcription and translation for driving expression of exogenous genes in eukaryotic cells, including a promoter, an enhancer, a transcription initiation region, a polyA processing and transcription termination signal, a ribosome binding region, a translation initiation signal, a translation termination signal and the like.
And (3) introducing the expression vector containing the BirA gene into host cells, and screening to obtain a Bir A cell strain which is stably expressed and has functional activity. The mode of introducing the expression vector into the cells comprises introducing the expression vector into mammalian cells by a chemical mode (cell transfection mediated by a microsphere structure taking lipid components such as liposome, lipofectamine and the like as a main body), a physical mode or a biological mode;
physical means of introducing the expression vector into a cell include, but are not limited to, introducing the DNA into a mammalian cell by electroporation;
the biological means for introducing the expression vector into the cell include, but are not limited to, the gene fragment being encapsulated into a viral particle, and the foreign gene fragment being introduced by means of infecting a mammalian cell with the viral particle;
the above host cells are cell lines commonly used for protein production; for example, HEK293F, Expi293 cells, CHO-K1, CHO-S cells, etc.
Screening of Positive cell lines
The screening of the above cells can be achieved by:
based on single cell separation technology such as flow cell sorting, high-expression cell strains can be selected by the expression of fluorescein genes (such as green, yellow and red fluorescent proteins GFP, YFP, RFP and the like) related to gene segments according to the fluorescence intensity;
the cells can be selected by expressing antibiotic resistance genes associated with gene segments in the expression vector and removing cells without gene integration or with relatively low expression by using antibiotics; high-expression cell strains can be selected by gradually increasing the concentration of antibiotics; obtaining the monoclonal cells by a limiting dilution method.
Construction of recombinant protein expression vector with Avi-His purification tag: adding an Avi-His purification tag at the N end or the C end of a target protein open reading frame of the protein expression vector to ensure that the target protein and the Avi-His tag are fused and expressed; the multifunctional fusion tag includes, but is not limited to, the purification tag His.
Functional characterization of Bir A cell lines
The functional activity of Bir a in Bir a overexpressing cell lines can be determined by:
in vivo biotinylated protein expression: transfecting the recombinant protein expression vector with the Avi-His purification tag into 293F-BirA cells, collecting the cells, and separating and purifying the protein;
and (3) detecting the biotinylation efficiency of the recombinant protein by adopting an ELISA method.
On the other hand, the cell line of the invention can be used for in vivo site-directed biotinylation in the process of recombinant protein production. The concrete application is as follows:
biotinylated recombinant protein: transfecting the recombinant protein expression vector with the Avi-His purification tag into a HEK293F-BirA cell, collecting the cell, and separating and purifying the protein; biotinylation is carried out by the kit of the in-vitro chemical labeling method of the non-biotinylated protein as a control; and detecting the biological activity of the biotinylated recombinant protein by using an ELISA method.
The invention is characterized in that: 1) the expression of the Bir A gene in eukaryotic mammalian cells is realized, and the Bir A gene is subjected to in-vivo site-specific biotinylation in the expression process of recombinant protein; 2) HEK293F widely used in recombinant protein production is selected as a host cell to overexpress Bir A, so that fixed-point biotinylation in cells is conveniently carried out while recombinant protein is produced, and the subsequent biological function detection efficiency is greatly improved; 3) provides a basic method and a technical approach for detecting and identifying in vivo biotinylated recombinant protein by using HEK293F-Bir A cell strain production.
A cell strain HEK293F-Bir A for stably expressing Bir A is preserved in China center for type culture Collection at 8 months and 4 days in 2020. The deposit unit code: CCTCC China center for type culture Collection; and (4) storage address: wuhan university in China; preservation state: survival; the preservation date is as follows: 8, month 4 in 2020; the preservation number is: CCTCC NO: C2020144; and (3) classification and naming: BirA overexpresses the human embryonic kidney cell line HEK 293F-BirA.
Description of the drawings:
figure 1 Bir a eukaryotic expression plasmid: bir A (UniProtKB-P06709(BIRA _ ECOLI)), a gene coding sequence (CDS), was inserted into the multicloning site of pcDNA3.1. Bir A expression is driven by a CMV promoter and a gene transcription regulation element thereof.
FIG. 2 expression vector containing Avi-His tag fusion protein: inserting a signal peptide, a recombinant protein gene and an Avi-his tag sequence into a eukaryotic expression vector; bringing them in the same Open Reading Frame (ORF); and ORF expression is driven by the EF1a promoter and its gene transcription regulatory elements.
FIG. 3 biotinylation function identification of HEK293F-Bir A stable expression cell strain: the recombinant proteins SLN8023 and SLN8024 can be combined with the coated SA, and the signal values show good concentration dependence, so that the results indicate that the SLN8023 and SLN8024 proteins have good biotinylation.
FIG. 4 site-directed biotinylated label detection in vitro and in vivo: the biotin-protein A and B of the recombinant protein biotinylated and marked in vitro have higher biotinylation efficiency than the in vivo site-directed biotinylation marked recombinant proteins Avi-protein A and Avi-protein B.
FIG. 5 detection of the biological activity of the in vitro and in vivo site-directed biotinylated recombinant proteins: the binding capacity of the in vivo site-directed biotinylation labeled recombinant proteins Avi-protein A and Avi-protein B and a receptor is obviously superior to that of in vitro biotinylation labeled recombinant proteins biotin-protein A and biotin-protein B.
Detailed Description
The present invention is further illustrated by the following specific examples.
The methods used in the following examples are conventional methods and some key reagents used in experiments unless otherwise specified.
Example 1 construction of recombinant eukaryotic expression vector pcDNA3.1-Bir A
Bir A (UniProtKB-P06709(BIR A _ ECOLI) is inserted into a multi-cloning site of a eukaryotic expression vector pcDNA3.1, the sequence translated into amino acid by the BirA gene is Seq 1, a CMV promoter and a gene transcription regulating element thereof drive the expression of Bir A in an Open Reading Frame (ORF) as shown in the attached figure 1 of the specification, the BirA gene is artificially synthesized after codon optimization, the synthesized BirA gene fragment and the pcDNA3.1 vector are subjected to Bmt I and Xba I double enzyme digestion, T4 ligase connection, DH 5 alpha competent cell transformation, amplification and plasmid extraction for later use, and a host cell is human HEK 293F.
Example 2 establishment of HEK293F-Bir A Stable cell line
HEK293F cells were cultured in Olympic (OPM-CD05, 81075-001) medium. Subculturing according to conventional method. The cells were cryopreserved in culture medium containing 10% DMSO, 1X 107Cells/ml per vial.
pcDNA3.1-Bir A transfection of HEK293F cells: day before transfection D0, inoculation 1.5X 106From/ml cells to 125ml cell shake flasks. Cells transfected with D1 after 24 hours: according to LipofectamineTM2000Transfection Reagent (Invitrogen, Cat #11668027), DNA LipofectamineTMMix at room temperature for 5min at 2000 ═ 1:5, and add to the cells.
Establishment of a cell line stably expressing HEK293-Bir A: d3, cells were passaged and pressure screened by adding 400ug/ml G418. D7, cell change fluid, G418400 ug/ml. D10, control cells all dead, HEK293F-Bir A cells were instead cultured in medium containing G418200 ug/ml. Carrying out conventional passage, amplification culture and liquid nitrogen preservation for later use.
Example 3 construction of protein expression vector containing Avi multifunctional Association tag and production of recombinant protein
Inserting a signal peptide, a recombinant protein gene and an avi-his tag sequence into a eukaryotic expression vector; the tag sequence of avi is translated into the amino acid sequence Seq 2; bringing them in the same Open Reading Frame (ORF); and ORF expression is driven by the EF1a promoter and its gene transcription regulatory elements. As shown in figure 2 of the specification. Transforming competent bacteria conventionally, amplifying, and extracting plasmid. HEK293F-BirA cells are transfected conventionally, the cells are amplified and cultured for 6 days, cell supernatants are collected by centrifugation, and the in-vivo fixed-point biotinylation recombinant proteins are purified and concentrated.
Example 4 in vivo biotinylation identification of recombinant protein production by cells stably expressing HEK293F-Bir A
The plate was coated with Streptavidin (Streptavidin, SA, 85878-5MG, Sigma) at 1ug/ml by conventional ELISA method overnight at 4 ℃; blocking with 1% BSA at room temperature for 1 hour; PBST is washed for 3 times, the proteins SLN8023 and SLN8024 are respectively diluted by 5ug by 5 times, and the mixture is incubated for 1 hour at room temperature; PBST was washed 3 times, and the secondary antibody anti-His-HRP (abcam, ab1187, 1:5000) was incubated at room temperature for 1 hour; PBST was washed 3 times, TMB was developed for 5 minutes, and after completion, OD was read at 450nm with a microplate reader. The GraphPad 8.0 software analyzed the experimental data. The experimental results are shown in the attached figure 3: the recombinant proteins SLN8023 and SLN8024 can be combined with the coated SA, and the signal values show good concentration dependence, so that the results indicate that the SLN8023 and SLN8024 proteins have good biotinylation.
Example 5 use of cells stably expressing HEK293F-Bir A for site-specific biotinylation in protein producing cells
Example 1: the biotinylation efficiency and biological activity of the recombinant protein A and the recombinant protein B were evaluated in vitro (biotin-protein A, biotin-protein B) and in vivo site-directed biotinylation of the recombinant protein A and the recombinant protein B (Avi-protein A, Avi-protein B) was produced using HEK293F-Bir A cells.
Coating a microporous plate with 1ug/ml protein according to a conventional ELISA method, and standing overnight at 4 ℃; blocking with 1% BSA at room temperature for 1 hour; PBST is washed for 3 times, each protein is diluted by 30ug and 3 times respectively, and the mixture is incubated for 1 hour at room temperature; PBST was washed 3 times and a secondary antibody SA-HRP (Sigma, S5512-.1MG, 1:5000) was incubated at room temperature for 1 hour; PBST was washed 3 times, TMB was developed for 5 minutes, and after completion, OD was read at 450nm with a microplate reader. The GraphPad 8.0 software analyzed the experimental data. The experimental results are shown in the attached figure 4: the biotin-protein A and B of the recombinant protein biotinylated and marked in vitro have higher biotinylation efficiency than the in vivo site-directed biotinylation marked recombinant proteins Avi-protein A and Avi-protein B.
In vitro and in vivo fixed point biotinylation labeled recombinant protein and receptor combination biological activity detection: the plate was coated with 1ug/ml SA (85878-5MG, Sigma) by conventional ELISA method overnight at 4 ℃; blocking with 1% BSA at room temperature for 1 hour; PBST is washed for 3 times, each recombinant protein is diluted by 30ug by 3 times, and the mixture is incubated for 1 hour at room temperature; PBST washing 3 times, receptor-mFc protein 1ug/ml, room temperature incubation for 1 hours; PBST was washed 3 times, and a secondary antibody anti-mFc-HRP (abcam, ab98717, 1:5000) was incubated at room temperature for 1 hour; PBST was washed 3 times, TMB was developed for 5 minutes, and after completion, OD was read at 450nm with a microplate reader. The GraphPad 8.0 software analyzed the experimental data. The experimental results are shown in the attached figure 5: the binding capacity of the in vivo site-directed biotinylation labeled recombinant proteins Avi-protein A and Avi-protein B and a receptor is obviously superior to that of in vitro biotinylation labeled recombinant proteins biotin-protein A and biotin-protein B.
Sequence listing
<110> Canuo (Shanghai) pharmaceutical science & technology Co., Ltd
<120> preparation method and application of cell strain for stably expressing biotin ligase Bir A
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 321
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> BirA amino acid sequence
<400> 1
Met Lys Asp Asn Thr Val Pro Leu Lys Leu Ile Ala Leu Leu Ala Asn
1 5 10 15
Gly Glu Phe His Ser Gly Glu Gln Leu Gly Glu Thr Leu Gly Met Ser
20 25 30
Arg Ala Ala Ile Asn Lys His Ile Gln Thr Leu Arg Asp Trp Gly Val
35 40 45
Asp Val Phe Thr Val Pro Gly Lys Gly Tyr Ser Leu Pro Glu Pro Ile
50 55 60
Gln Leu Leu Asn Ala Lys Gln Ile Leu Gly Gln Leu Asp Gly Gly Ser
65 70 75 80
Val Ala Val Leu Pro Val Ile Asp Ser Thr Asn Gln Tyr Leu Leu Asp
85 90 95
Arg Ile Gly Glu Leu Lys Ser Gly Asp Ala Cys Ile Ala Glu Tyr Gln
100 105 110
Gln Ala Gly Arg Gly Arg Arg Gly Arg Lys Trp Phe Ser Pro Phe Gly
115 120 125
Ala Asn Leu Tyr Leu Ser Met Phe Trp Arg Leu Glu Gln Gly Pro Ala
130 135 140
Ala Ala Ile Gly Leu Ser Leu Val Ile Gly Ile Val Met Ala Glu Val
145 150 155 160
Leu Arg Lys Leu Gly Ala Asp Lys Val Arg Val Lys Trp Pro Asn Asp
165 170 175
Leu Tyr Leu Gln Asp Arg Lys Leu Ala Gly Ile Leu Val Glu Leu Thr
180 185 190
Gly Lys Thr Gly Asp Ala Ala Gln Ile Val Ile Gly Ala Gly Ile Asn
195 200 205
Met Ala Met Arg Arg Val Glu Glu Ser Val Val Asn Gln Gly Trp Ile
210 215 220
Thr Leu Gln Glu Ala Gly Ile Asn Leu Asp Arg Asn Thr Leu Ala Ala
225 230 235 240
Met Leu Ile Arg Glu Leu Arg Ala Ala Leu Glu Leu Phe Glu Gln Glu
245 250 255
Gly Leu Ala Pro Tyr Leu Ser Arg Trp Glu Lys Leu Asp Asn Phe Ile
260 265 270
Asn Arg Pro Val Lys Leu Ile Ile Gly Asp Lys Glu Ile Phe Gly Ile
275 280 285
Ser Arg Gly Ile Asp Lys Gln Gly Ala Leu Leu Leu Glu Gln Asp Gly
290 295 300
Ile Ile Lys Pro Trp Met Gly Gly Glu Ile Ser Leu Arg Ser Ala Glu
305 310 315 320
Lys
<210> 2
<211> 15
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<220>
<223> Avi-tag amino acid sequence
<400> 2
Gly Leu Asn Asp Ile Phe Glu Ala Gln Lys Ile Glu Trp His Glu
1 5 10 15

Claims (6)

1. A eukaryotic expression cell line stably expressing Bir A with functional activity.
2. The cell line of claim 1, which is obtained by inserting a foreign gene fragment into the genome of the cell by genetic engineering means.
3. The cell line of claim 1 which is HEK293F or other cell line useful for recombinant protein production.
4. The use of the cell of claim 1 for in vivo site-directed biotinylation during recombinant protein production.
5. The recombinant protein expression vector of claim 4, which is a multifunctional combination tag expression vector containing an Avi-protein purification tag.
6. The method for detecting the recombinant protein according to claim 5, which comprises ELISA, western-blot, or the like.
CN202110288528.3A 2021-03-17 2021-03-17 Preparation method and application of cell strain for stably expressing biotin ligase Bir A Pending CN113061589A (en)

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CN111100199A (en) * 2018-12-29 2020-05-05 北京百普赛斯生物科技有限公司 Fluorescein labeled protein tetramer and preparation method and application thereof

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KATRIN HOFFMANN ET AL: "A platform for discovery of functional cell-penetrating peptides for efficient multi-cargo intracellular delivery", SCI REP., vol. 8, no. 1, pages 9 - 12 *
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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN118207171A (en) * 2024-05-21 2024-06-18 北京量维生物科技研究院有限公司 Biotin ligase mutant and its use in biotin production

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