CN118308393A - Expression system of escherichia coli without antibiotic resistance marker, construction method and application thereof - Google Patents
Expression system of escherichia coli without antibiotic resistance marker, construction method and application thereof Download PDFInfo
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Abstract
The invention discloses an expression system of escherichia coli without antibiotic resistance markers, and a construction method and application thereof, and belongs to the technical field of bioengineering. The expression system takes sfp genes as screening marks, the antibiotic screening genes are removed from the escherichia coli expression system, antibiotics are not required to be added in the recombinant protein expression process to provide selection pressure, the transmission of the antibiotic genes in the environment and organisms is avoided, and stable passage of expression plasmids in host strains is ensured. After removal of the antibiotic gene, the burden on the host cell is reduced. The method simultaneously optimizes the mode of expressing the recombinant protein by using the escherichia coli, does not need to add an inducer in the expression process of the recombinant protein, and is a technical progress in the field of escherichia coli expression systems for producing the recombinant protein.
Description
Technical Field
The invention relates to the technical field of bioengineering, in particular to an expression system of escherichia coli without antibiotic resistance markers, and a construction method and application thereof.
Background
Coli has the advantages of fast growth speed, low culture cost, easy genetic operation, good productivity and perfect fermentation process, and becomes one of the most perfect recombinant protein production factories. Currently, there are a number of molecular tools and corresponding recombinant expression protocols available for high level production of foreign proteins, including high level expression plasmids and corresponding engineered strains and various culture strategies. From the theoretical point of view, the target gene is cloned into any available expression vector, transformed into a selected host, and induced to express, thus obtaining the recombinant protein. Most expression vectors contain antibiotic genes as selectable markers, so antibiotics are often used as selectable pressures in the production of recombinant proteins by E.coli to improve the stability of plasmid-carrying cultures. However, the use of antibiotics not only increases the production cost, but also brings potential risks of residue of antibiotics in recombinant proteins, transmission of antibiotic resistance genes, and the like. In addition, the presence of the antibiotic resistance gene may also affect the amount of expression of the protein of interest, with the expression of the antibiotic gene imposing an additional burden on the host during fermentation. The development of stable expression vectors that do not require resistance selection markers has therefore become an important development in the production of recombinant proteins.
Disclosure of Invention
The invention aims to provide an expression system of escherichia coli without antibiotic resistance markers, and a construction method and application thereof, so as to solve the problems in the prior art.
In order to achieve the above object, the present invention provides the following solutions:
The invention provides a construction method of an expression system of escherichia coli without antibiotic resistance markers, wherein the expression system takes sfp genes as screening markers.
Preferably, the method comprises the following steps:
(1) Constructing a gene deletion mutant strain of the escherichia coli acpS;
(2) Constructing an expression vector taking sfp gene as a screening marker;
(3) Transforming the expression vector in the step (2) into the E.coli acpS gene deletion mutant strain in the step (1), namely the E.coli expression system without antibiotic resistance mark.
Preferably, the construction of the E.coli acpS gene deletion mutant specifically comprises:
Selecting 500bp sequences on the upstream and downstream of acpS genes as homologous arms at two ends, and inserting a fragment FRT-Cm R -FRT on a pKD3 plasmid in the middle to be used as a targeting fragment for knocking out acpS genes;
And (3) performing heat shock transformation on the plasmid pRP101-sfp and pKD46 to obtain escherichia coli shock competent cells, performing PCR amplification on the target fragment of the knocked-out acpS gene, performing shock transformation on the shock competent cells, and knocking out acpS genes, namely obtaining the escherichia coli acpS gene deletion mutant.
Preferably, the expression vector is pET28b-P1-Kan R:: sfp.
Preferably, the E.coli expression system without antibiotic resistance marker is E.coli BL21 (DE 3) delta acpS (pET 28b-P1-Kan R:: sfp).
The invention also provides an expression system of the escherichia coli without antibiotic resistance markers, which is obtained by the construction method.
The invention also provides application of sfp gene in preparing an expression vector of escherichia coli without antibiotic resistance marker.
The invention also provides an expression vector, which takes sfp genes as screening markers.
Preferably, the expression vector further comprises a nucleotide sequence encoding recombinant protein YebF-Metch.
The invention also provides a construction method of the expression vector pET28b-P1-Kan R:sfp:
designing a primer, cloning a nucleotide sequence for encoding an Sfp amino acid sequence, performing enzyme digestion on a connecting vector pET28b-P1, replacing a kanamycin resistance gene Kan R of a primary vector with an Sfp gene, and constructing a vector pET28b-P1-Kan R capable of expressing a secretory protein.
The invention also provides an application of the expression system or the expression vector in preparation of recombinant proteins.
The invention also provides an expression method of the recombinant protein without the resistance screening mark, and the recombinant protein is obtained by using the expression system.
Based on the technical scheme, the invention has the following technical effects:
(1) The invention removes antibiotic screening genes in an escherichia coli expression system, avoids the transmission of the antibiotic genes in the environment and organisms, and ensures that the expression plasmid can be stably passaged in host strains. After removal of the antibiotic gene, the burden on the host cell is reduced.
(2) The method optimizes the mode of expressing recombinant protein by using escherichia coli, does not need to add antibiotics in the expression process to provide selection pressure, avoids the abuse of antibiotics and the generation of drug resistance, and is a technical progress in the field of escherichia coli expression systems for producing recombinant protein.
(3) The lacI gene of the original vector is knocked out in the process of constructing the expression vector, the target protein is secreted outside cells of escherichia coli by using the guide protein YebF (YebF-target protein), an inducer IPTG is not required to be added in the expression process, the recombinant protein secretion table is successfully reached to the culture medium supernatant, the downstream protein purification step is simplified, the production cost is saved, and the recombinant protein secretion table has great market value and application prospect.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are needed in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and other drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 is a schematic diagram of vector pRP 101-sfp;
FIG. 2 shows red single colonies of E.coli BL21 (DE 3) containing plasmids pRP101-sfp and pKD46 (temperature sensitive plasmid) grown on solid medium;
FIG. 3 shows the construction method of E.coli BL21 (DE 3) Δ acpS mutant;
FIG. 4 is a schematic diagram of vector pET28b-P1-Kan R::sfp-FRT-GmR -FRT;
FIG. 5 shows the result of SDS-PAGE analysis of the vector pET28b-P1-Kan R in strain E.coli BL21 (DE 3) delta acpS; m: GS PRESTAINED Protein Ladder (10-180 kDa); lane 1: pET28b-P1 induces expression in E.coli BL21 (DE 3) for 24h to collect culture medium supernatant; lane 2: pET28b-P1-Kan R sfp was expressed in E.coli BL21 (DE 3) delta acpS for 24h of the collected culture supernatant;
FIG. 6 shows the result of SDS-PAGE analysis of expression vector pET28b-P1-Kan R:. Sfp in E.coli BL21 (DE 3) Δ acpS for 20 th generation of secretion expression recombinant protein; m: GS PRESTAINED Protein Ladder (10-180 kDa); lane 1: pET28b-P1-Kan R:: sfp (E.coli BL21 (DE 3) Δ acpS) represents the medium supernatant collected for 24h initially; lane 2: pET28b-P1-Kan R:: culture supernatant collected from 20 th generation expression of sfp (E.coli BL21 (DE 3) Δ acpS).
Detailed Description
The present invention will be described in detail with reference to specific examples. These embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art.
The technical means used in the examples are conventional means well known to those skilled in the art unless otherwise indicated. The test methods in the following examples are conventional methods unless otherwise specified. The reagents and materials employed, unless otherwise indicated, are commercially available.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the present invention. The preferred methods and materials described herein are presented for illustrative purposes only.
The practice of the present invention will employ, unless otherwise indicated, techniques of microbiology, tissue culture, molecular biology, chemistry, biochemistry, DNA recombination and bioinformatics, which are apparent to those skilled in the art. These techniques are fully explained in the published literature, and the DNA extraction, gene editing method and construction of the gene editing vector used in the present invention can be carried out by the methods disclosed in the prior art except for the methods used in the examples described below.
The terms "nucleic acid", "nucleic acid sequence", "nucleotide", "nucleic acid molecule" or "polynucleotide" as used herein are meant to include isolated DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., messenger RNA), natural types, mutant types, synthetic DNA or RNA molecules, DNA or RNA molecules composed of nucleotide analogs, single-or double-stranded structures. Such nucleic acids or polynucleotides include, but are not limited to, gene coding sequences, antisense sequences, and regulatory sequences of non-coding regions. These terms include a gene. "Gene" or "gene sequence" is used broadly to refer to a functional DNA nucleic acid sequence. Thus, a gene may include introns and exons in genomic sequences, and/or coding sequences in cDNA, and/or cDNA and regulatory sequences thereof.
In addition, in order to more intuitively understand the technical scheme of the present invention, some terms related to the present invention are explained as follows:
"mutant" refers to a mutant individual having a phenotype that differs from the wild type.
The expression vector refers to a vector which is formed by adding expression elements (such as a promoter, RBS, terminator and the like) on the basis of the basic skeleton of a cloning vector so that a target gene can be expressed.
The technical scheme of the invention is conventional in the field, and the reagents or raw materials are purchased from commercial sources or are disclosed.
The embodiment of the invention provides a construction method of an expression system of escherichia coli without antibiotic resistance markers, wherein the expression system takes sfp genes as screening markers.
Preferably, the method comprises the following steps:
(1) Constructing a gene deletion mutant strain of the escherichia coli acpS;
(2) Constructing an expression vector taking sfp gene as a screening marker;
(3) Transforming the expression vector in the step (2) into the E.coli acpS gene deletion mutant strain in the step (1), namely the E.coli expression system without antibiotic resistance mark.
The embodiment of the invention provides an expression system of escherichia coli without antibiotic resistance markers, which is obtained by the construction method.
In some specific embodiments, the expression system is obtained by transforming a recombinant expression vector into a corresponding host strain, preferably E.coli, more preferably a acpS-knocked-out E.coli mutant BL21 (DE 3) Δ acpS.
The embodiment of the invention provides application of sfp genes in preparation of an expression vector of escherichia coli without antibiotic resistance markers.
The embodiment of the invention provides an expression vector, which takes sfp genes as screening markers.
In some specific embodiments, the expression vector is preferably an engineered pET series vector.
The embodiment of the invention provides a construction method of the expression vector pET28b-P1-Kan R:
designing a primer, cloning a nucleotide sequence for encoding an Sfp amino acid sequence, performing enzyme digestion on a connecting vector pET28b-P1, replacing a kanamycin resistance gene Kan R of a primary vector with an Sfp gene, and constructing a vector pET28b-P1-Kan R capable of expressing a secretory protein.
The embodiment of the invention provides an application of the expression system or the expression vector in preparation of recombinant proteins.
The embodiment of the invention provides a recombinant protein expression method without a resistance screening mark, and the recombinant protein is obtained by using the expression system.
In some specific embodiments, the method comprises the steps of:
Coli BL21 (DE 3) delta acpS containing vector pET28b-P1-Kan R:: sfp was cultured for 24 hours, and the supernatant was collected by centrifugation to obtain a culture medium supernatant containing the target protein.
The expression conditions are preferably 30 ℃ for 24 hours;
The rotating speed of the constant temperature oscillator is 200rpm;
The conditions for collecting the supernatant by centrifugation are preferably 12000rpm, and the centrifugation is carried out at 4 ℃ for 10min
Example 1
Construction of the expression System of coli BL21 (DE 3) Delta acpS (pET 28b-P1-Kan R:: sfp)
(1) Construction of the E.coli BL21 (DE 3) Δ acpS mutant Strain
Knocking out acpS genes in E.coli genome by using Red homologous recombination method, wherein the nucleotide sequence of acpS genes is shown as SEQ ID NO. 1:
SEQ ID NO.1：ATGGCAATATTAGGTTTAGGCACGGATATTGTGGAGATCGCTC GCATCGAAGCGGTGATCGCCCGATCCGGTGATCGCCTGGCACGCCGCGTATTAAGCGATAACGAATGGGCTATCTGGAAAACGCACCACCAGCCGGTGCGTTTTCTGGCGAAGCGTTTTGCTGTGAAAGAAGCCGCAGCAAAAGCGTTTGGCACCGGGATCCGCAATGGTCTGGCGTTTAATCAATTTGAAGTATTCAATGATGAGCTCGGCAAACCACGGCTACGGCTATGGGGCGAGGCATTAAAACTGGCGGAAAAGCTGGGCGTTGCAAATATGCATGTAACGCTGGCAGATGAGCGGCACTATGCTTGTGCCACGGTAATTATTGAAAGTTAA.
The 500bp sequences on the upstream and downstream of acpS gene are selected as homologous arms at two ends, and the fragment FRT-Cm R -FRT on the pKD3 plasmid is inserted in the middle to be used as a targeting fragment for knocking out acpS gene.
Plasmid pRP101-sfp (shown in FIG. 1) was heat-shock transformed into E.coli BL21 (DE 3) competent cells with pKD46, and E.coli shock competent cells were prepared by overnight culture and selection of red single colonies (shown in FIG. 2). Plasmid pRP101-sfp is a temperature sensitive plasmid, and can be lost by culturing at 42 ℃, and is provided with a spectinomycin resistance gene Spec R; also contains gene sfp, and the expressed protein can replace the function of AcpS; meanwhile, the red fluorescent protein is expressed and can be used as a screening mark. PCR greatly amplifying the target fragment of acpS knocked out genes, converting the prepared electric shock competent cells by electric shock, and selecting red single colonies for colony PCR identification. The acpS gene is knocked out (shown in figure 3), and the gene is successfully prepared into escherichia coli heat shock competent cells.
(2) Construction of pET28b-P1-Kan R::sfp-FRT-GmR -FRT intermediate expression vector
The sfp gene was ligated with the FRT-Gm R -FRT fragment using overlap PCR, and a pET28b-P1-Kan R::sfp-FRT-GmR -FRT expression vector was constructed by a seamless cloning method, in which the sfp-FRT-Gm R -FRT sequence replaced the kanamycin resistance gene in the original vector, as shown in FIG. 4.
(3) Construction of a resistance-free selection marker expression vector pET28b-P1-Kan R:: sfp
E.coli BL21 (DE 3) delta acpS is prepared into heat shock competent cells after the acpS gene is knocked out successfully, pET28b-P1-Kan R is converted into sfp-FRT-GmR-FRT intermediate expression vectors, single colonies are selected and cultured at 42 ℃ in an oscillating way, and E.coli BL21 (DE 3) delta acpS (pET 28b-P1-Kan R::sfp-FRT-GmR -FRT) white single colonies which successfully eliminate pRP101-sfp plasmids are selected.
The cell is prepared into a heat shock competent cell, the pCP20 plasmid is transformed and induced to express, the resistance gene is eliminated, and finally, an expression system without a resistance screening marker of E.coli BL21 (DE 3) delta acpS (pET 28b-P1-Kan R:: sfp) is successfully constructed.
Example 2
Expression of recombinant proteins in resistance-free selectable marker expression systems
E.coli BL21 (DE 3) Delta acpS containing vector pET28b-P1-Kan R:: sfp was cultured at 30℃and 200rpm for 24 hours, and the supernatant was collected by centrifugation at 12000rpm and 4℃for 10 minutes.
As shown in FIG. 5, sfp can secrete and express recombinant protein YebF-Metch in E.coli BL21 (DE 3) delta acpS, antibiotics and inducer IPTG are not needed in the expression process, and the secretory expression quantity of pET28b-P1 in E.coli BL21 (DE 3) is consistent.
The amino acid sequence of the recombinant protein (YebF-Metch) is shown in SEQ ID NO. 2:
SEQ ID NO.2：ANNETSKSVTFPKCEGLDAAGIAASVKRDYQQNRVARWADDQ KIVGQADPVAWVSLQDIQGKDDKWSVPLTVRGKSADIHYQVSVDCKAGMAEYQRR PASGGGGAGSEFENLYFQHRHQGPIFDTRPSPFNPNQPRPGPIY.
example 3
Vector pET28b-P1-Kan R. Sfp is stably transferred in E.coli BL21 (DE 3) delta acpS
E.coli BL21 (DE 3) delta acpS containing vector pET28b-P1-Kan R:. Sfp was streaked on LB solid medium, cultured for 12h at 37℃for one generation, then subsequently transferred for 20 generations, expressed recombinant protein YebF-Metch h at 30℃at 200rpm, and the supernatant was collected by centrifugation at 12000rpm for 10min at 4 ℃.
As shown in the result of analysis of recombinant protein YebF-Metch by SDS-PAGE, the vector pET28b-P1-Kan R shows that sfp can stably substitute in E.coli BL21 (DE 3) delta acpS, and the secretion expression level of the recombinant protein is not obviously changed.
In E.coli, the holo-ACP synthase AcpS transfers coenzyme A4' -phosphopantetheine to an Acyl Carrier Protein (ACP), a key component in fatty acid synthesis, and this post-translational modification converts ACP from an inactive form to an active form of holo-ACP. acpS is the deletion of a lethal gene, sfp in bacillus subtilis can replace acpS's function in escherichia coli. The invention constructs an expression vector without a resistance screening marker by utilizing the complementary characteristics of functions of AcpS and Sfp.
The amino acid sequence of Sfp is shown as SEQ ID NO. 3:
SEQ ID NO.3：MGGKIYGIYMDRPLSQEENERFMSFISPEKREKCRRFYHKEDA HRTLLGDVLVRSVISRQYQLDKSDIRFSTQEYGKPCIPDLPDAHFNISHSGRWVICAFDSQPIGIDIEKTKPISLEIAKRFFSKTEYSDLLAKDKDEQTDYFYHLWSMKESFIKQEGKGLSLPLDSFSVRLHQDGQVSIELPDSHSPCYIKTYEVDPGYKMAVCAVHPDFPEDITMVSYEELL.
At present, the successful cases of realizing the extracellular secretion expression of the recombinant protein based on a non-resistance screening marker vector in escherichia coli are few, and the reported extracellular secretion expression level of the recombinant protein is low, and has a larger distance from the industrialization requirement. The invention also realizes the high-level secretory expression of the recombinant protein without the addition of the inducer.
It is to be understood that the above examples of the present invention are provided for clarity of illustration only and are not limiting of the embodiments of the present invention. Other variations or modifications of the above description will be apparent to persons of ordinary skill in the art. It is not necessary here nor is it exhaustive of all embodiments. Any modification, equivalent replacement, improvement, etc. which come within the spirit and principles of the invention are desired to be protected by the following claims.
Claims (10)
1. A method for constructing an expression system of escherichia coli without antibiotic resistance markers, which is characterized in that the expression system takes sfp genes as screening markers.
2. The method of construction according to claim 1, comprising the steps of:
(1) Constructing a gene deletion mutant strain of the escherichia coli acpS;
(2) Constructing an expression vector taking sfp gene as a screening marker;
(3) Transforming the expression vector in the step (2) into the E.coli acpS gene deletion mutant strain in the step (1), namely the E.coli expression system without antibiotic resistance mark.
3. The construction method according to claim 2, wherein the construction of the escherichia coli acpS gene deletion mutant is specifically:
Selecting 500bp sequences on the upstream and downstream of acpS genes as homologous arms at two ends, and inserting a fragment FRT-Cm R -FRT on a pKD3 plasmid in the middle to be used as a targeting fragment for knocking out acpS genes;
And (3) performing heat shock transformation on the plasmid pRP101-sfp and pKD46 to obtain escherichia coli shock competent cells, performing PCR amplification on the target fragment of the knocked-out acpS gene, performing shock transformation on the shock competent cells, and knocking out acpS genes, namely obtaining the escherichia coli acpS gene deletion mutant.
4. The construction method according to claim 2, wherein the expression vector is pET28b-P1-Kan R:: sfp.
5. The method according to claim 2, wherein the E.coli antibiotic resistance marker-free expression system is E.coli BL21 (DE 3) Δ acpS (pET 28b-P1-Kan R:: sfp).
6. An expression system of E.coli without antibiotic resistance marker obtained by the construction method according to any one of claims 1 to 5.
Application of acpS gene in preparing expression vector of colibacillus without antibiotic resistance mark.
8. An expression vector comprising sfp gene as a selectable marker.
9. Use of the expression system of claim 6 or the expression vector of claim 8 for the preparation of recombinant proteins.
10. A method for expressing a recombinant protein without a resistance selection marker, wherein the recombinant protein is obtained by using the expression system according to claim 6.
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|---|---|---|---|---|
| CN102604983A (en) * | 2012-03-08 | 2012-07-25 | 刘紫琦 | Construction method of gene engineering strain without plasmid and antibiotic resistance screening marker |
| CN104531725A (en) * | 2014-12-23 | 2015-04-22 | 华南农业大学 | Sequence capable of being modified by 4'-phosphopantetheine and method thereof for immobilizing protein |
| CN105132349A (en) * | 2015-09-09 | 2015-12-09 | 中国科学院微生物研究所 | Recombinant cell screening system and building method thereof |
| CN116064363A (en) * | 2023-02-07 | 2023-05-05 | 徐州合谷生命科技有限公司 | Genetic engineering strain for producing blue-looking and application thereof |
| CN117070538A (en) * | 2023-08-16 | 2023-11-17 | 浙江工业大学 | Application of ppt1 gene as screening marker in screening of auxotrophs |
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Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102604983A (en) * | 2012-03-08 | 2012-07-25 | 刘紫琦 | Construction method of gene engineering strain without plasmid and antibiotic resistance screening marker |
| CN104531725A (en) * | 2014-12-23 | 2015-04-22 | 华南农业大学 | Sequence capable of being modified by 4'-phosphopantetheine and method thereof for immobilizing protein |
| CN105132349A (en) * | 2015-09-09 | 2015-12-09 | 中国科学院微生物研究所 | Recombinant cell screening system and building method thereof |
| CN116064363A (en) * | 2023-02-07 | 2023-05-05 | 徐州合谷生命科技有限公司 | Genetic engineering strain for producing blue-looking and application thereof |
| CN117070538A (en) * | 2023-08-16 | 2023-11-17 | 浙江工业大学 | Application of ppt1 gene as screening marker in screening of auxotrophs |
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