CN111196842A - Expression and purification method of non-transmembrane structural domain of outer membrane transport channel protein - Google Patents
Expression and purification method of non-transmembrane structural domain of outer membrane transport channel protein Download PDFInfo
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- CN111196842A CN111196842A CN202010021615.8A CN202010021615A CN111196842A CN 111196842 A CN111196842 A CN 111196842A CN 202010021615 A CN202010021615 A CN 202010021615A CN 111196842 A CN111196842 A CN 111196842A
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Abstract
The invention discloses an expression and purification method of a non-transmembrane domain of an outer membrane transport channel protein, belonging to the technical field of biology. According to the invention, the non-transmembrane domain of the outer membrane transport channel protein is truncated, and an amino acid sequence of 30-273 of BamA-POTRA is selected as a truncation body, which contains 3 POTRAs and covers the main functional region of the non-transmembrane domain of the BamA protein; moreover, the gene sequence coding the truncation body is connected with pET-28a plasmid and carries out prokaryotic expression, a large amount of high-purity target protein can be expressed in vitro, the in vitro expression efficiency of the non-transmembrane structural domain of the BamA protein is greatly improved, and the purity of the expressed target protein is high and can reach more than 90 percent; the invention has very important significance for analyzing the three-dimensional structure of the BamA protein through the in vitro expression of the high-purity BamA-POTRA truncated protein.
Description
Technical Field
The invention relates to the technical field of biology, in particular to an expression and purification method of a non-transmembrane domain of an outer membrane transport channel protein.
Background
The transmembrane outer membrane protein is almost β -barrel structure, generally directly called OMPs (outer membrane protein), the OMPs are β -barrel structure proteins formed by 8-24 even number antiparallel β -folds through adjacent hydrogen bonds, the β -fold is mainly a double-nature structure formed by hydrophobic amino acids facing membrane phospholipid and hydrophilic amino acids facing the inner layer of the barrel structure, and each β -fold is connected by a long ring facing the cell outside and a short ring facing the periplasmic space.
BamA (also called Omp85) is the most important core component in the BAM complex, has high conservation and the deletion of the BamA is lethal, BamA consists of a β -barrel domain integrated to the outer membrane at the C-terminal end and 5 POTRA (Polypeptide transported-Associated) domains facing the periplasmic space at the N-terminal end, the POTRA domains belong to the non-transmembrane domains of the outer membrane Transport channel protein, and the connection structures between the POTRAs have strong flexibility, so that the POTRAs have mobility and extensibility, can be used as binding sites of BamA and other monomers of the BAM complex, and can be specifically bound with OMPs in an unfolded state through the interaction between β -sheet structures.
In recent years, functional studies on the BamA protein have been greatly advanced, but the non-transmembrane domain of the BamA protein is expressed in vitro with very low efficiency and is difficult to purify, which has been a difficulty that restricts the structural analysis of the BamA protein.
Disclosure of Invention
In view of the above prior art, the present invention aims to provide a method for expression and purification of the non-transmembrane domain of the outer membrane transport channel protein. According to the invention, by constructing the truncation of the Borrelia burgdorferi BamA-POTRA, the in-vitro expression efficiency of the non-transmembrane domain of the BamA protein is greatly improved, and the expressed target protein has high purity, so that the structural analysis of the BamA protein is facilitated.
In order to achieve the purpose, the invention adopts the following technical scheme:
in a first aspect of the present invention, there is provided a method for purifying the expression of a non-transmembrane domain of an outer membrane transport channel protein, comprising the steps of:
(1) performing PCR amplification by using the BamA gene as a template and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers to obtain a BamA-POTRA truncation gene;
(2) connecting the BamA-POTRA truncated gene obtained by amplification in the step (1) into an expression vector to obtain a recombinant expression vector, then transferring the recombinant expression vector into a prokaryotic expression strain to obtain engineering bacteria, culturing the engineering bacteria until the value of a bacterial liquid A600OD is 0.6-0.8, adding an inducer, and continuing culturing for 16-18 h;
(3) taking a bacterial liquid which is added with an inducer and cultured, centrifugally collecting thalli, and suspending the thalli by using a bacterium collecting buffer solution; breaking cells of the thalli, centrifuging and collecting supernatant; and (3) carrying out primary separation on the supernatant by using a nickel ion affinity chromatography column, carrying out sample loading and elution by using heparin affinity chromatography, and collecting an eluent sample in a peak range to obtain the purified BamA-POTRA truncated body protein.
Preferably, in step (1), the PCR amplification conditions are: denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 32 cycles; finally, keeping the temperature at 72 ℃ for 10 min.
Preferably, in the step (1), the nucleotide sequence of the BamA-POTRA truncation gene is shown as SEQ ID NO. 3.
Preferably, in step (2), the expression vector is a PET-28a plasmid. A plasmid is a small circular DNA molecule, and is usually used for carrying a foreign gene, introducing the foreign gene into a host cell, and expressing a protein. The types of plasmids are diverse, and the origin of replication, replication ability, selection for resistance, and compatibility with other plasmids are different for different plasmids. Therefore, even if the same foreign gene fragment is ligated to different types of plasmids and prokaryotic expression is performed, the expression effect is greatly different. Experiments show that the PET-28a plasmid is used as an expression vector, and the expression effect on the BamA-POTRA truncated body protein is optimal.
Preferably, in the step (2), the prokaryotic expression strain is BL21(DE 3).
Preferably, in step (2), the inducer is isopropyl- β -D-thiogalactoside (IPTG) and the final concentration of the inducer after addition is 0.5 mM.
Preferably, in the step (3), the bacteria collecting buffer contains 200mM NaCl, 20mM hydroxyethylpiperazineethiosulfonic acid (HepeS); the pH value of the bacterium collecting buffer solution is 7.0.
Preferably, in the step (3), the conditions for performing the primary separation on the nickel ion affinity chromatography column on the supernatant are as follows: eluting with 25mM and 50mM imidazole respectively; the buffer loading was 20mM Hepes, 200mM NaCl, pH 7.0; the elution buffer was 20mM Hepes, 1M NaCl, pH 7.0.
Preferably, in the step (3), the amino acid sequence of the BamA-POTRA truncation protein is shown in SEQ ID NO. 4.
In a second aspect of the present invention, there is provided a use of the BamA-POTRA truncation protein prepared by the above expression and purification method for structural analysis of the BamA protein.
The invention has the beneficial effects that:
according to the invention, the non-transmembrane domain of the outer membrane transport channel protein is truncated, so that the in vitro expression efficiency of the non-transmembrane domain of the BamA protein is greatly improved, and the purity of the expressed target protein is high and can reach more than 90%; the invention has very important significance for analyzing the three-dimensional structure of the BamA protein through the in vitro expression of the high-purity BamA-POTRA truncated protein.
Drawings
FIG. 1: electrophorogram of the protein expressed in example 1 of the present invention; wherein, M is protein Maker, 1 is sample before induction, and 2 is sample after induction.
FIG. 2: SDS-PAGE picture of protein nickel column affinity chromatography expressed in the embodiment 1 of the invention; wherein, M: protein maker, 1: and (3) crushing bacterial liquid, centrifuging to obtain precipitate, 2: and (3) centrifuging after crushing the bacterial liquid to obtain a supernatant, 3: flow-through before treatment with imidazole, 4: media before treatment with imidazole, 5: flow-through after washing with 25mM imidazole, 6: medium after washing with 25mM imidazole, 7: flow-through after washing with 50mM imidazole, 8: 50mM imidazole-washed media.
FIG. 3: HiTrap TM Heparin HP map of the protein expressed in example 1 of the present invention.
FIG. 4: the HiTrap TM Heparin HP of FIG. 3 was sampled electrophoretogram corresponding to the peak position (SDS-PAGE).
FIG. 5: SDS-PAGE picture of nickel column affinity chromatography of protein expressed in comparative example 1; wherein, M: protein maker, 1: and (3) crushing bacterial liquid, centrifuging to obtain precipitate, 2: and (3) centrifuging after crushing the bacterial liquid to obtain a supernatant, 3: flow-through before treatment with imidazole, 4: media before treatment with imidazole, 5: flow-through after washing with 25mM imidazole, 6: medium after washing with 25mM imidazole, 7: flow-through after washing with 50mM imidazole, 8: 50mM imidazole-washed media.
FIG. 6: SDS-PAGE picture of nickel column affinity chromatography of protein expressed in comparative example 2; wherein, M: protein maker, 1: and (3) crushing bacterial liquid, centrifuging to obtain precipitate, 2: and (3) centrifuging after crushing the bacterial liquid to obtain a supernatant, 3: flow-through before treatment with imidazole, 4: media before treatment with imidazole, 5: flow-through after washing with 25mM imidazole, 6: medium after washing with 25mM imidazole, 7: flow-through after washing with 50mM imidazole, 8: 50mM imidazole-washed media.
FIG. 7: SDS-PAGE picture of nickel column affinity chromatography of protein expressed in comparative example 3; wherein, M: protein maker, 1: and (3) crushing bacterial liquid, centrifuging to obtain precipitate, 2: and (3) centrifuging after crushing the bacterial liquid to obtain a supernatant, 3: flow-through before treatment with imidazole, 4: media before treatment with imidazole, 5: flow-through after washing with 25mM imidazole, 6: medium after washing with 25mM imidazole, 7: flow-through after washing with 50mM imidazole, 8: 50mM imidazole-washed media.
FIG. 8: HiTrap TM Heparin HP profiles of the proteins expressed in comparative example 3 of the present invention.
Detailed Description
It should be noted that the following detailed description is exemplary and is intended to provide further explanation of the disclosure. 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 to which this application belongs.
As described in the background section, the efficiency of expression of the non-transmembrane domain of the BamA protein in vitro is very low, and the difficulty of purification is a difficult point to restrict the structural analysis of the BamA protein.
Based on this, the object of the present invention is to provide a method capable of expressing a non-transmembrane domain of a BamA protein with high efficiency in vitro.
Because the entire non-transmembrane domain of the BamA protein (labeled as BamA-POTRA, or BamA30-432, the amino acid sequence of which is shown in SEQ ID NO. 5) is expressed very inefficiently in vitro, the present invention contemplates truncation of the non-transmembrane domain of the BamA protein. The whole non-transmembrane domain of the BamA protein consists of 403 amino acids, and how to select the truncation is the key technical point of the invention, and the selected truncation is required to cover the main functional region of the non-transmembrane domain of the BamA protein on one hand and ensure that the selected truncation can be efficiently expressed in vitro on the other hand. According to the invention, a plurality of groups of truncation bodies with different lengths are designed according to structure prediction data, gene segments for coding the truncation bodies with different lengths are connected with various expression vectors, and the prokaryotic expression condition of the truncation bodies in vitro is inspected.
As a result, the 30-273 amino acid sequence (SEQ ID NO.4) of BamA-POTRA was selected as a truncated body, which contains 3 POTRAs and covers the main functional region of the non-transmembrane domain of BamA protein; moreover, the gene sequence (SEQ ID NO.3) encoding the truncation is connected with pET-28a plasmid and prokaryotic expression is carried out, so that a large amount of high-purity target protein can be expressed in vitro. The present invention has been made by the fact that the non-transmembrane domain of the BamA protein can be structurally resolved by the expressed high purity target protein.
In order to make the technical solutions of the present application more clearly understood by those skilled in the art, the technical solutions of the present application will be described in detail below with reference to specific embodiments.
The test materials used in the examples of the present invention are all conventional in the art and commercially available. The experimental procedures, for which no detailed conditions are indicated, were carried out according to the usual experimental procedures or according to the instructions recommended by the supplier.
Example 1:
(1) according to the structure prediction data of the BamA protein, the following primers are designed:
primer 1:5 '-CGGGATCCAAAATCATTAAAGGT-3'; (SEQ ID NO.1)
Primer 2:5 '-CCCTCGAGTTATTCGCTCAGAAAG-3'; (SEQ ID NO. 2).
Using BamA gene as a template, and using a primer 1 and a primer 2 to carry out PCR amplification, wherein the conditions of the PCR amplification are as follows: denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 32 cycles; finally, keeping the temperature at 72 ℃ for 10 min.
After the PCR product is preliminarily confirmed by 1% agarose electrophoresis, gel cutting and recovery are carried out, and the nucleotide sequence of the PCR product is shown as SEQ ID NO.3 through sequencing.
(2) Connecting the PET-28a cloning vector with a PCR product, wherein a connection reaction system is as follows:
the ligation reaction system is subjected to ligation reaction at 16 ℃ for 4-6 h.
(3) Transforming E.coli DH5 α competent cells with the ligation products, coating LB plate (final concentration of kanamycin is 50 mug/ml) containing kanamycin, selecting positive clones, extracting plasmid DNA by using a plasmid miniprep kit, sequencing the plasmid DNA, comparing the sequencing result with the published sequence of a gene bank (genbank), and confirming that the gene sequence is correct, thus constructing the recombinant expression vector.
(4) The constructed recombinant expression vector with successful sequencing is transformed into a prokaryotic expression strain BL21(DE3), an LB plate containing kanamycin (the final concentration of the kanamycin is 50 mu g/ml) is coated, a single clone is selected and inoculated into a 5ml LB (containing kanamycin) test tube, shaking culture is carried out at 37 ℃ at 220r, and the culture is terminated when the value of a test bacterium liquid A600OD is 0.6-0.8.
5ml of the above-mentioned cultured bacterial solution was put into a flask containing 800ml of LB medium (containing 50. mu.g/ml kanamycin) and cultured in a shaker at 37 ℃ to measure the A600OD value of the bacterial solution and 0.6-0.8 OD value, IPTG (isopropyl- β -D-thiogalactoside) was added to the bacterial solution to induce a final concentration of 0.5mM, and the culture was continued for 16 hours.
(5) The bacteria were collected, centrifuged at 6000rpm at 4 ℃ for 15 minutes, the supernatant removed and the pellet collected. The pellet was suspended with pH7.0, 200mM NaCl, 20mM HepeS harvest buffer.
The bacteria are lysed by a high pressure disruption method, then centrifuged for 30min at 4 ℃ at 12,000r/min, the supernatant after disruption and centrifugation is equilibrated for more than two hours by nickel column affinity chromatography, the impure proteins are washed off by 25mM and 50mM imidazole respectively, and the samples are respectively taken for SDS-PAGE analysis. As shown in FIG. 2, the medium after washing with 50mM imidazole contained a large amount of the target protein.
Then eluting the target protein with 500mM imidazole (containing 350mM NaCl), centrifuging and concentrating the eluted target protein at 4 ℃ for 12,000r/min, continuously changing the solution with low salt, finally concentrating to 2ml with the salt concentration of 200mM NaCl, passing through a heparin column, and carrying out heparin affinity chromatography, wherein a single peak appears, and the peak value is high and broad, as shown in figure 3. The positions of the peaks were sampled and electrophoresed (SDS-PAGE), and as can be seen in FIG. 4, the purified protein was single and large in amount, and the purity of the protein could reach more than 90% by SDS-PAGE electrophoresis.
The expressed and purified target protein is a BamA-POTRA truncated protein which is marked as BamA-POTRA30-273-pET28 a.
Comparative example 1:
the gene (shown in SEQ ID NO. 6) of the whole non-transmembrane domain of the coding BamA protein is connected to a plasmid pET-28a, the recombinant expression vector is transformed into a prokaryotic expression strain BL21(DE3) by the method of example 1, and the expression and purification are carried out, and samples are taken for electrophoresis detection after nickel column affinity chromatography.
The results are shown in FIG. 5, and it can be seen from FIG. 5 that most of the target proteins are in the precipitate, which is an inclusion body, and influence the subsequent further experiments.
Comparative example 2:
the gene (shown in SEQ ID NO. 6) of the whole non-transmembrane domain of the BamA protein is connected to a plasmid pET-28bs (the pET-28bs contains a soluble SUMO label which can promote the solubility of the target protein in the supernatant), the recombinant expression vector is transformed into a prokaryotic expression strain BL21(DE3) by the method of example 1, expression and purification are carried out, and samples are sampled for electrophoretic detection after nickel column affinity chromatography.
The results are shown in FIG. 6, and it can be seen from FIG. 6 that most of the target protein is still in the precipitate, which is an inclusion body, and affects the subsequent further experiments.
Comparative example 3:
the gene fragment of the amino acid segment (corresponding to the 1 st to 352 nd amino acids in the amino acid sequence shown in SEQ ID NO. 5) of the non-transmembrane domain 30-352 of the BamA protein is connected to a plasmid pET-28a, the recombinant expression vector is transformed into a prokaryotic expression strain BL21(DE3) by the method of example 1, and the prokaryotic expression strain is expressed and purified, and is sampled for electrophoretic detection after nickel column affinity chromatography.
As shown in FIG. 7, the target protein of the truncated form was improved to some extent in FIG. 7, and the medium after washing with 50mM imidazole contained a certain amount of target protein, and the target protein was eluted with 500mM imidazole (containing 350mM NaCl) and passed through a heparin column, as shown in FIG. 8, the peak was large and small, indicating that the protein was not pure and the yield was low.
The above description is only a preferred embodiment of the present application and is not intended to limit the present application, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, improvement and the like made within the spirit and principle of the present application shall be included in the protection scope of the present application.
SEQUENCE LISTING
<110> university of Jinan
<120> expression and purification method of non-transmembrane domain of outer membrane transport channel protein
<130>2020
<160>6
<170>PatentIn version 3.5
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Claims (10)
1. A method for purifying the expression of the non-transmembrane domain of an outer membrane transport channel protein, comprising the steps of:
(1) performing PCR amplification by using the BamA gene as a template and the sequences shown in SEQ ID NO.1 and SEQ ID NO.2 as primers to obtain a BamA-POTRA truncation gene;
(2) connecting the BamA-POTRA truncated gene obtained by amplification in the step (1) into an expression vector to obtain a recombinant expression vector, then transferring the recombinant expression vector into a prokaryotic expression strain to obtain engineering bacteria, culturing the engineering bacteria until the value of a bacterial liquid A600OD is 0.6-0.8, adding an inducer, and continuing culturing for 16-18 h;
(3) taking a bacterial liquid which is added with an inducer and cultured, centrifugally collecting thalli, and suspending the thalli by using a bacterium collecting buffer solution; breaking cells of the thalli, centrifuging and collecting supernatant; and (3) carrying out primary separation on the supernatant by using a nickel ion affinity chromatography column, carrying out sample loading and elution by using heparin affinity chromatography, and collecting an eluent sample in a peak range to obtain the purified BamA-POTRA truncated body protein.
2. The method for purifying expression according to claim 1, wherein in the step (1), the PCR amplification conditions are: denaturation at 94 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 1min for 32 cycles; finally, keeping the temperature at 72 ℃ for 10 min.
3. The method for purifying expression according to claim 1, wherein in the step (1), the nucleotide sequence of the BamA-POTRA truncation gene is represented by SEQ ID No. 3.
4. The method for purifying expression according to claim 1, wherein in the step (2), the expression vector is a PET-28a plasmid.
5. The expression purification method according to claim 1, wherein in step (2), the prokaryotic expression strain is BL21(DE 3).
6. The method for purifying expression according to claim 1, wherein in the step (2), the inducer is IPTG, and the final concentration of the inducer after addition is 0.5 mM.
7. The method for purifying expression according to claim 1, wherein in the step (3), the bacteria-collecting buffer contains 200mM NaCl, 20mM HepeS; the pH value of the bacterium collecting buffer solution is 7.0.
8. The expression purification method according to claim 1, wherein in the step (3), the conditions for subjecting the supernatant to primary separation by a nickel ion affinity chromatography column are as follows: eluting with 25mM and 50mM imidazole respectively; the buffer loading was 20mM Hepes, 200mM NaCl, pH 7.0; the elution buffer was 20mM Hepes, 1M NaCl, pH 7.0.
9. The expression purification method according to claim 1, wherein in the step (3), the amino acid sequence of the BamA-POTRA truncation protein is shown in SEQ ID NO. 4.
10. Use of a BamA-POTRA truncation protein produced by the expression purification method according to any one of claims 1 to 9 for structural analysis of the BamA protein.
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| CN103108652A (en) * | 2010-05-14 | 2013-05-15 | 巴克斯特国际公司 | Chimeric ospa genes,proteins, and methods of use thereof |
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| CN1159831A (en) * | 1994-08-17 | 1997-09-17 | 马克斯·普朗克促进科学协会 | Vaccines containing borrelia burgdorferi OSPG |
| CN103108652A (en) * | 2010-05-14 | 2013-05-15 | 巴克斯特国际公司 | Chimeric ospa genes,proteins, and methods of use thereof |
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