CN117737103A - Cell surface display method utilizing animal defensin to fuse heterologous protein - Google Patents
Cell surface display method utilizing animal defensin to fuse heterologous protein Download PDFInfo
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Abstract
The invention provides a cell surface display method for fusing animal defensin with heterologous protein, which connects rhesus monkey leukocyte theta-defensin monomer or multimer with the heterologous protein through a joint, induces expression in host bacteria, and realizes the display of the heterologous protein on the surface of the host bacteria. The invention has simple operation and good display effect, does not need to use a protein transport system of host bacteria, and is a general surface display technology of a prokaryotic/eukaryotic system. The fusion protein of the invention has simple design and preparation, can induce expression by a conventional method, and does not need complex molecular operation. The heterologous protein has good display effect, is tightly combined with cell membranes, can be used as a whole-cell catalyst by host cells, can be continuously and repeatedly used for many times, has good activity, and solves the technical problems that the free enzyme reaction is difficult to recover and has high cost.
Description
Technical Field
The invention belongs to the technical field of bioengineering, and particularly relates to a technology for displaying heterologous proteins on the surfaces of escherichia coli and pichia pastoris cells.
Background
Display technology for anchoring heterologous proteins on the cell surface is widely used in the fields of drug screening, biocatalysis, biosensors, etc. as it can be applied to almost all types of organisms (Park, m. Surface display technology for biosensor applications: a review.2020). The core of the technology is to fuse the target protein gene sequence with a specific carrier gene sequence, introduce the target protein gene sequence into a receptor cell, and express and anchor the target protein on the cell surface by utilizing a protein transport mechanism in the receptor cell. Commonly used E.coli transporters include ice nucleoprotein and S-layer proteins (bloom et al decoding microbes: surface display of proteins on Escherichia coll.2011). However, the general surface display technology for prokaryotic/eukaryotic systems has not been developed due to the highly receptor cell-dependent protein transport mechanisms.
Defensins are cationic antibacterial peptides rich in disulfide bonds, are widely distributed in fungi, plants and animals, and are important regulatory molecules with direct sterilization function in biological immune systems. The defensin sterilization mechanism is mainly related to the cell membrane structure of microorganisms: positively charged defensin molecules or multimers thereof can interact with negatively charged phospholipids and water molecules on cell membranes to increase the permeability of the biofilm, resulting in leakage of the cell contents. By mimicking this property of defensins, some peptide polymers have been developed which are very effective in killing drug resistant bacteria (Zhang et al host defense peptide mimicking cyclic peptoid polymers exerting strong activity against drug-resistance bacteria 2022). There is no report on the application of defensins in cell surface display technology.
Disclosure of Invention
The first object of the present invention is to provide a cell surface display method using animal defensin fusion heterologous proteins, which can display heterologous proteins on the surface of prokaryotic and eukaryotic cells without the aid of host cell protein transport system, and can reduce host energy and metabolic burden.
The second object of the invention is to provide the application of animal defensin in cell surface display technology, and the application of the defensin and the electrostatic interaction of cell membranes is utilized to realize cell surface display, so that the method is a surface display method which is universal for prokaryotic and eukaryotic systems.
In order to achieve the above purpose, the invention provides a cell surface display method utilizing animal defensin fusion heterologous proteins, which connects rhesus monkey leukocyte theta-defensin monomer or multimer with the heterologous proteins through a joint, induces expression in host bacteria, and realizes the display of the heterologous proteins on the surface of the host bacteria.
As a preferred embodiment, the host bacterium is E.coli or Pichia pastoris. The surface display of the protein is realized by utilizing the electrostatic action of defensins and cell membranes, and the protein is not dependent on the protein transport mechanism of receptor cells, and is generally used for a prokaryotic/eukaryotic system. The preferred embodiment of the present invention selects E.coli and Pichia pastoris as hosts, as they are the most commonly used prokaryotic and eukaryotic protein expression systems, respectively. In fact, bacillus subtilis, lactic acid bacteria, saccharomyces cerevisiae, etc. may be used as host bacteria.
As a preferred embodiment, the rhesus leukocyte θ -defensin multimer has a degree of aggregation of 2 to 6. Rhesus leukocyte θ -defensin multimers refer to multiple repeat rhesus leukocyte θ -defensin amino acid sequences.
As a preferred embodiment, the joint comprises one or more sections of rigid joint or flexible joint. The linker refers to an amino acid chain which plays a role in connection between two fusion proteins, and has certain flexibility to allow the proteins at two sides to complete independent functions.
As a preferred embodiment, the linker is one or more of EAAAK, APAPAPAPAPAPAP, AAYAAY, PTPPTTPTPPTTPTPTP, GGGGS (SEQ ID NO. 1-SEQ ID NO. 5). The number of the joints may be single or stacked, and in the preferred embodiment of the present invention, 1 to 6 joints are used.
As a preferred embodiment, the rhesus leukocyte θ -defensin monomer or multimer is linked to the C-terminus of a heterologous protein. The N-terminus of the heterologous protein may contain a signal peptide or precursor peptide that will be cleaved, and is therefore typically selected for attachment to the C-terminus of the protein.
To achieve the second object of the invention, the invention discloses the application of animal defensin in cell surface display technology, wherein the animal defensin is rhesus leukocyte theta-defensin.
As a preferred embodiment, the cells are E.coli or Pichia cells.
The invention has the advantages that the invention provides a general technology for realizing the display of the heterologous protein on the surfaces of the prokaryotic and eukaryotic cells without the help of a host cell protein transport system, and simultaneously, the invention can reduce the host energy and metabolism burden. The fusion protein is simple in design and preparation, can be induced to express by a conventional method, and does not need complex molecular operation. The heterologous protein has good display effect, is tightly combined with cell membranes, can be used as a whole-cell catalyst by host cells, can be continuously and repeatedly used for many times, has good activity, and solves the technical problems that the free enzyme reaction is difficult to recover and has high cost.
Drawings
FIG. 1 is a laser confocal microscopy image of E.coli with surface displaying jellyfish green fluorescent protein.
Fig. 2 is a pichia pastoris laser confocal microscope image with the surface displaying jellyfish green fluorescent protein.
FIG. 3 is an SDS-PAGE electrophoresis of E.coli surface displaying polyphosphate kinase.
FIG. 4 is an SDS-PAGE electrophoresis of E.coli surface displaying nucleoside kinase.
FIG. 5 is a graph showing the results of multi-round catalysis of whole cells of E.coli.
Detailed Description
The technical solutions in the embodiments of the present invention are clearly and completely described below in connection with specific embodiments. The test methods used in the examples described below are conventional methods unless otherwise specified, and materials, reagents, etc. used are commercially available. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention.
Example 1: jellyfish green fluorescent protein displayed on surface of escherichia coli
Nucleotide sequence of 3-segment rhesus theta-defensin (GFCRCLCRRGVCRCICTR) 3 (SEQ ID NO. 6) the gene was ligated to the pET28a expression vector by ligating the nucleotide sequence C-terminus of the jellyfish green fluorescent protein through 3-segment EAAAK rigid linker, transformed into E.coli BL21 (DE 3), the obtained positive clone was inoculated into 50mL of LB containing 50mg/L kanamycin, cultured at 37℃at 220rpm for 6-8 hours, then inoculated into 100mL of LB at 37℃in an inoculum size of 2% and left to stand OD 600 When the concentration reaches 0.6-0.8, IPTG with the final concentration of 0.2mM is added, the temperature is 18 ℃, and 220rpm is used for induction for 16-18 hours. The cells were collected by centrifugation, the supernatant was discarded, and an appropriate amount of PB buffer (pH 6.0) was added to resuspend the cells. Crushing the resuspended thallus with a high-pressure crusher under 600-700bar, centrifuging to collect precipitate, cleaning the precipitate with sterile water for multiple times, and observing with a laser confocal microscope. As a result, as shown in FIG. 1, the precipitate was seen to emit clear green fluorescence, indicating that the green fluorescent protein was displayed on the E.coli surface.
Example 2: jellyfish green fluorescent protein displayed on pichia pastoris surface
Nucleotide sequence of 3-segment rhesus theta-defensin (GFCRCLCRRGVCRCICTR) 3 Connecting the nucleotide sequence C end of jellyfish green fluorescent protein with 3 segments of EAAAK rigid joints, connecting the gene to pPICC 3.5K expression vector, transforming to Pichia pastoris GS115, connecting the obtained positive clone to 25mL BMGY, culturing at 30 ℃ at 250rpm for overnight, centrifuging to collect cells, discarding the supernatant, and re-suspending the cells to OD by BMMY 600 =1.0, methanol was added every 24h to a final concentration of 0.5%, and expression was induced for 96h. The cells were collected by centrifugation, the supernatant was discarded, and an appropriate amount of PB buffer (pH 6.0) was added to resuspend the cells. Cells were observed using a laser confocal microscope. As a result, it can be seen that green fluorescence was emitted from the cell membrane, indicating that the green fluorescent protein was displayed on the Pichia pastoris surface, as shown in FIG. 2.
Example 3: surface display of Escherichia coli polyphosphate kinase
Will 1Nucleotide sequence of rhesus theta-defensin 3 th segment (GFCRCLCRRGVCRCICTR) 3 The gene is connected to a pET28a expression vector through a 1-3 segment EAAAK rigid linker and the C end of a nucleotide sequence of Sulfurovum lithotrophicum source polyphosphate kinase, and is transformed into escherichia coli BL21 (DE 3), and the obtained positive clones are named as Sl-1, sl-2 and Sl-3 respectively. The three bacteria were inoculated into 50mL of LB containing 50mg/L kanamycin, cultured at 37℃for 6-8 hours at 220rpm, then inoculated into 100mL of LB at 2% inoculum size, cultured at 37℃until OD 600 When the concentration reaches 0.6-0.8, IPTG with the final concentration of 0.2mM is added, the temperature is 18 ℃, and 220rpm is used for induction for 16-18 hours. The cells were collected by centrifugation, the supernatant was discarded, and an appropriate amount of PB buffer (pH 6.0) was added to resuspend the cells. The resuspended cells were crushed in a high-pressure crusher at 600-700bar, and the pellet and supernatant were collected by centrifugation, and SDS-PAGE analysis was performed on the two fractions, the results of which are shown in FIG. 3. Wherein lane 1 is a protein marker; lane 2 is Sl-1 crushed supernatant; lane 3 is Sl-1 cell debris; lane 4 is Sl-2 crushed supernatant; lane 5 is Sl-2 cell debris; lane 6 is Sl-3 crushed supernatant; lane 7 is Sl-3 cell debris. Most of the target protein can be seen in the cell debris, indicating that the polyphosphate kinase is displayed on the E.coli surface.
Example 4: surface display nucleoside kinase of escherichia coli
Nucleotide sequence of 3-segment rhesus theta-defensin (GFCRCLCRRGVCRCICTR) 3 The gene is connected to a pET28a expression vector through a 3-segment EAAAK rigid joint and the C end of a nucleotide sequence of a nucleotide kinase derived from Phorcysiothermogenesis, and is transformed into escherichia coli BL21 (DE 3), and the obtained positive clone is named PtNK-RTD. Inoculating into 50mL LB containing 50mg/L kanamycin, culturing at 37deg.C and 220rpm for 6-8 hr, inoculating into 100mL LB at 2% inoculum size, culturing at 37deg.C, and standing for OD 600 When the concentration reaches 0.6-0.8, IPTG with the final concentration of 0.2mM is added, the temperature is 18 ℃, and 220rpm is used for induction for 16-18 hours. The cells were collected by centrifugation, the supernatant was discarded, and an appropriate amount of PB buffer (pH 6.0) was added to resuspend the cells. Crushing the resuspended thallus in high-pressure crusher under 600-700bar, centrifuging to collect precipitate and supernatant, and subjecting the two components to SDS-PAGE analysis, the results are shown in FIG. 4. Wherein lane 1 is a protein marker; lane 2 PtNK-RTD disruption supernatant; lane 3 is PtNK-RTD cell debris. Most of the target protein can be seen in cell debris, indicating that nucleoside kinase is displayed on the E.coli surface.
Example 5: surface display cells as whole cell catalysts
The reaction system comprises 200mM cytidine, 5mM ATP,40mM MgCl 2 ,50mM polyP 6 3-5mL of 50mM PB buffer solution, 30mL of total volume of each of the Sl-3 and PtNK-RTD cell debris resuspension, 40-55 ℃ and pH 5.0-6.0, and the reaction time is 240min, thereby obtaining the cytidylic acid product. Centrifuging at 8000rpm for 5min, and adding the precipitate into a new reaction system for 240min. Repeated 6 times.
The yield of cytidine acid was measured by high performance liquid chromatography according to the catalytic system of example 5. As shown in FIG. 5, the yield of cytidine acid in the 6 th reaction is still maintained above 80%.
The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.
Claims (8)
1. A cell surface display method utilizing animal defensin to fuse heterologous protein is characterized in that a rhesus monkey leukocyte theta-defensin monomer or multimer is connected with the heterologous protein through a joint, and induced to be expressed in host bacteria, so that the heterologous protein is displayed on the surface of the host bacteria.
2. The method for displaying a cell surface by using an animal defensin fusion heterologous protein according to claim 1, wherein the host bacterium is escherichia coli or pichia pastoris.
3. The method for displaying a cell surface using animal defensin fusion heterologous protein according to claim 1, wherein the rhesus leukocyte θ -defensin multimer has a degree of aggregation of 2-6.
4. The method of claim 1, wherein the linker comprises one or more rigid or flexible linkers.
5. The method for displaying a cell surface using an animal defensin fusion heterologous protein according to claim 1 wherein the linker is one or more of EAAAK, APAPAPAPAPAPAP, AAYAAY, PTPPTTPTPPTTPTPTP, GGGGS.
6. The method for displaying a cell surface using an animal defensin fusion heterologous protein according to claim 1 wherein the rhesus leukocyte θ -defensin monomer or multimer is linked to the C-terminus of the heterologous protein.
7. The application of animal defensin in cell surface display technology is characterized in that the animal defensin is rhesus leukocyte theta-defensin.
8. The use of animal defensin according to claim 7 wherein the cell is an e.
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