CN114574512B - Preparation of cell-penetrating peptide-target protein complex and method for efficiently introducing cell-penetrating peptide-target protein complex into streptomycete living cells - Google Patents
Preparation of cell-penetrating peptide-target protein complex and method for efficiently introducing cell-penetrating peptide-target protein complex into streptomycete living cells Download PDFInfo
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/70—Vectors or expression systems specially adapted for E. coli
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- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
- C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
- C12N15/76—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora for Actinomyces; for Streptomyces
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- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/10—Fusion polypeptide containing a localisation/targetting motif containing a tag for extracellular membrane crossing, e.g. TAT or VP22
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- C12N2800/00—Nucleic acids vectors
- C12N2800/10—Plasmid DNA
- C12N2800/101—Plasmid DNA for bacteria
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- Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
- Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
- Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
- Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
- Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change
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Abstract
The invention discloses a preparation method of a cell membrane penetrating peptide-target protein compound and a method for efficiently introducing the cell penetrating peptide-target protein compound into a streptomycete living cell. The method can help the target exogenous protein to efficiently cross the complex cell wall and cell membrane barrier of the gram-positive bacteria, and is favorable for mediating the exogenous protein to efficiently enter the streptomycete living cells by the method so as to study the living cell level or the visual level of cell gene expression regulation, metabolite production monitoring and the like.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a preparation method of a cell membrane-penetrating peptide-target protein compound and a method for efficiently introducing the compound into streptomycete living cells.
Background
Cell penetrating peptides (Cell penetrating peptide, CPPs) are a class of short peptides consisting of about 5 to 30 amino acids that can be used as a living cell substance delivery system to carry proteins, nucleic acids, nanomaterials, etc. of interest through the physiological barrier cell membrane of animal cells by covalent or non-covalent coupling and have less effect on cell activity at certain concentrations. Currently, CPPs have been used more widely in non-invasive drug delivery tools targeting intracellular molecules. Recent researches show that CPP can not only carry oligonucleotides, small interfering RNA, plasmids, nano materials and the like to overcome animal cell membranes, but also carry the small interfering RNA or plasmids and the like into higher plant, algae and microorganism cells containing cell wall structures, but related reports are less common.
At present, related researches on the transportation of 'cargoes' to living cells of microorganisms are focused on the fact that the penetrating peptide can increase the uptake of antimicrobial active substances such as antimicrobial peptides, peptide nucleic acids and functional photodynamic antimicrobial nano particles by the cells of the microorganisms, so that the antibacterial effect is enhanced.
In addition, in recent years, researchers have also begun to apply transmembrane peptides to microbial cell carriage of plasmids and nanomaterials. In 2018, munish Kumar realizes the transport of escherichia coli and staphylococcus aureus of the Tat-based derivative transmembrane peptide and nano-gold conjugate for the first time. In 2019, md Monirul Islam et al first passed through the 205 and kb plasmid pMSR227 containing the fluorescent protein mCherry reporter gene with a transmembrane peptide (KH) 9 The BP100 was directly incubated with cells after noncovalent coupling, successfully transformed into E.coli DH 5. Alpha. Cells, and the transformation efficiency of the method was confirmed to be similar to that of electroporation and significantly higher than that of the heat shock method.
In summary, it can be seen that the transmembrane peptide has begun to be applied to live cell carriage of foreign substances by microbial cells, which has the potential to carry various foreign substances into live cells of microorganisms. However, at present, related researches and application reports of the penetrating peptide for mediating the foreign protein to enter the streptomycete living cells are not seen.
Disclosure of Invention
The invention aims to provide a method for efficiently introducing exogenous proteins into streptomycete living cells, and a preparation method of a membrane penetrating peptide-target protein complex related to the method. The target protein is introduced into the streptomycete living cells by using the membrane penetrating peptide, and can be applied to the aspects of streptomycete metabolite detection, gene expression regulation, cell function regulation, biological control and the like.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a preparation method of a cell membrane penetrating peptide-target protein complex is characterized by comprising the following steps: the method comprises the following steps:
(1) Constructing a CPP-mCherry protein expression plasmid pET28a-CPP-mCherry: constructing a membrane penetrating peptide, 4 glycine connecting peptides and fluorescent protein mCherry fusion expression gene in an escherichia coli expression plasmid pET-28a (+);
(2) Transformation of plasmid pET28a-CPP-mCherry into E.coli by chloride-calcium transformationE.coliBL21 (DE 3) competent cells;
(3) Inoculating CPP-mCherry recombinant expression strain into 5mL LB liquid medium containing kanamycin, activating at 37deg.C and 220 rpm for 8 h, inoculating the activated strain into 250 mL 2 XYT medium at a volume ratio of 1:100, adding 250 μL kanamycin (concentration 50-55 mg/mL), culturing for 4.5-5.5 h, and standing for OD 600 The value reaches 0.6-0.8, the bacterial liquid is cooled to 16 ℃, IPTG is added, and induced expression is carried out for 16-20 h at 16 ℃ and 220 rpm;
(4) CPP-mCherry fusion protein purification and concentration: centrifuging the bacterial liquid for induction expression, collecting bacterial cells, adding 30mL of PBS solution into the bacterial cells, peeling the bacterial cells from the wall of a centrifugal bottle by using a glass rod, thoroughly re-suspending the bacterial cells on a vortex instrument (the bacterial cells obtained by 500mL of bacterial liquid are dissolved in 30mL of PBS at most), ultrasonically crushing the re-suspended bacterial cells, transferring the crushed bacterial liquid into a 50 mL centrifuge tube, balancing, centrifuging at 4 ℃ and 10000 rpm for 30 min, and collecting the supernatant; after the cobalt affinity chromatography column is balanced for 3-5 column volumes by PBS, passing the collected supernatant through the column, and repeating column hanging for one time; washing 3-5 column volumes with PBS for removing impurities such as cell debris which cannot be removed by high-speed centrifugation; washing 3-5 column volumes with PBS containing 5 mM imidazole for removing non-specifically bound hybrid proteins from the gel; washing 5-8 column volumes with PBS containing 150 mM imidazole to dissociate target proteins specifically bound to the gel, and collecting all eluates; washing 3-5 column volumes with PBS containing 500 mM imidazole, dialyzing the collected protein, and concentrating to obtain cell penetrating peptide-target protein recombinant fusion protein.
(5) Cobalt affinity chromatography column renaturation: first using ddH 2 O-washing 6-8 column volumes, followed by 2 column volumes with 0.4N NaOH, immediately with ddH 2 O-washing 6-8 column volumes followed by 0.1M EDTA wash column with ddH 2 Immediately after O-washing 6-8 column volumes, the column was washed with 0.1M CoCl 2 Washing 5-8 column volumes and incubating for 15 min at room temperature; incubation was completed with a large amount of ddH 2 After O washing, 3-5 column volumes are balanced by PBS, and the product can be stored for a long time at 4 ℃.
Further, the transmembrane peptide and the amino acid sequence thereof in the step (1) are one of the following:
Tat：YGRKKRRQRRR；
ANTP：RQIKIWFQNRRMKWKK；
KFF 3 K：KFFKFFKFFK；
R 9 ：RRRRRRRRR；
K 9 ：KKKKKKKKK；
KH 9 -BP100：KHKHKHKHKHKHKHKHKHKKLFKKILKYL；
the cell penetrating peptide-target protein complex obtained in the step (4) is Tat-mCherry, ANTP-mCherry, (KFF) 3 K-mCherry、R 9 -mCherry、K 9 -mCherry、(KH) 9 -one of BP 100-mCherry.
In the step (3), the kanamycin concentration in the LB liquid medium is 50-55 mug/mL, and the IPTG final concentration is 0.1-0.4 mM.
In the step (4), the ultrasonic breaking time of the re-suspended thalli is 15 min, the ultrasonic is carried out for 2s, the stop is carried out for 4s, and the ultrasonic process is operated in a dark environment.
In the step (4), the specific steps of protein dialysis and concentration are as follows: placing the collected proteins into a dialysis bag, immersing the dialysis bag in PBS solution to replace imidazole in the protein collection liquid, and placing the dialysis device at 4 ℃ overnight in a dark place because the target proteins have fluorescence; changing the dialysate once in the next day; placing the dialyzed protein into a protein concentration tube, centrifuging at 4 ℃ and 4000 g, and concentrating the protein to 1.5-2 mL with the concentration of about 150 mu M; the whole experimental operation is protected from light; the obtained protein was stored at-80℃in a freeze-protected manner.
A high-efficiency streptomycete living cell exogenous protein introducing method comprises the following steps:
(1) Treatment of Streptomyces spores: adding 4 mL PBS solution on a streptomycete flat plate, then lightly scraping the streptomycete spores by an inoculating loop, blowing and uniformly mixing by a pipetting gun, uniformly dividing bacterial liquid into a centrifuge tube, centrifuging at 4000 rpm for 3 min, collecting thalli, washing for 2 times by PBS, and sucking out redundant solution to obtain streptomycete sporophytes;
(2) Taking the same Streptomyces sporophytes, and adding Tat-mCherry, ANTP-mCherry, (KFF) respectively 3 K-mCherry、K 9 -mCherry、R 9 -mCherry、(KH) 9 Uniformly diluting BP100-mCherry to 50 mu M by using PBS, taking 100 mu L of diluent, adding the diluent into cells, gently mixing, incubating at 34 ℃ in the dark for 1 h, and taking 50 mu M fluorescent protein mCherry without a penetrating peptide segment as a control;
(3) After incubation, centrifuging at 10000 rpm for 1 min, removing the supernatant, adding 500 mu L of PBS, re-suspending and washing once, centrifuging, removing the supernatant, adding 100 mu L of 0.25% trypsin, and digesting at 37 ℃ for 3-5 min to remove proteins adsorbed on the surface of the thalli; after centrifugation to remove trypsin and washing twice with PBS, the cells were resuspended in 500. Mu.L of PBS.
The method provided by the invention realizes the high-efficiency loading of the exogenous protein into the streptomycete living cells, which is helpful for realizing the researches of physiological condition research on the streptomycete metabolic pathway by taking the protein as an element, intervention regulation and control on the streptomycete gene expression at the living cell level, metabolite detection at the living cell level, fluorescent marker visualization and the like.
The invention can realize the purpose of efficiently transferring the exogenous target protein to the streptomycete living cells, and has no obvious influence on the cell activity. The method can help the target exogenous protein to efficiently cross the complex cell wall and cell membrane barrier of the gram-positive bacteria, and is favorable for mediating the exogenous protein to efficiently enter the streptomycete living cells by the method so as to study the living cell level or the visual level of cell gene expression regulation, metabolite production monitoring and the like.
Drawings
FIG. 1 is a schematic diagram of the introduction of exogenous target proteins into Streptomyces living cells mediated by a transmembrane peptide of the present invention.
FIG. 2 is a graph showing fluorescence intensity of 50. Mu.M different transmembrane peptide-mCherry fusion proteins in live Streptomyces cells in step (4) of example 2.
FIG. 3 is a graph showing the statistics of fluorescence intensities of 50. Mu.M different transmembrane peptide-mCherry fusion proteins in live Streptomyces cells in step (5) of example 2.
FIG. 4 is a graph showing the statistics of the cell entry efficiency of 50. Mu.M different transmembrane peptide-mCherry fusion proteins in live Streptomyces cells in step (5) of example 2.
FIG. 5 shows the concentration of the peptide (KH) in step (5) of example 2 of the present invention 9 Statistics of fluorescence intensity of BP100-mCherry fusion protein in Streptomyces living cells.
FIG. 6 shows the concentration of the peptide (KH) in step (5) of example 2 of the present invention 9 -statistics of the cell entry efficiency of BP100-mCherry fusion protein in Streptomyces living cells.
FIG. 7 shows the concentration of the peptide (KH) in step (5) of example 2 of the present invention 9 Effect of BP100-mCherry fusion protein on streptomyces cell activity.
Detailed Description
A method for efficiently introducing exogenous proteins into live Streptomyces cells and verification of the carrying effect thereof, and a method for preparing fusion proteins by the method are described in detail below by means of specific examples. Unless otherwise indicated, the reagents and experimental methods referred to below are conventional in the art.
Example 1
The preparation method of the cell penetrating peptide-target protein complex comprises the following steps:
(1) Constructing a CPP-mCherry protein expression plasmid pET28a-CPP-mCherry: constructing a penetrating peptide, 4 glycine connecting peptides and fluorescent protein mCherry fusion expression gene in an escherichia coli expression plasmid pET-28a (+), wherein the penetrating peptide and the amino acid sequence thereof are Tat (YGRKKRRQRRR), ANTP (RQIKIWFQNRRMKWKK) and KFF 3 K（KFFKFFKFFK）、R 9 （RRRRRRRRR）、K 9 （KKKKKKKKK）、KH 9 -BP100(KHKHKHKHKHKHKHKHKHKKLFKKILKYL)。
(2) Transformation of plasmid pET28a-CPP-mCherry into E.coli by chloride-calcium transformationE.coliBL21 (DE 3) competent cells.
(3) CPP-mCherry recombinant expression strain was inoculated into 5mL LB liquid medium containing 50. Mu.g/mL Kanamycin (Kanamycin, kan), activated 8 h at 37℃and 220 rpm, the activated strain was inoculated into 250 mL 2 XYT medium at a volume ratio of 1:100, and after 250. Mu.L Kan was added, the strain was cultured for about 5 h until OD was obtained 600 The value reaches about 0.6 to 0.8, the bacterial liquid is cooled to 16 ℃, IPTG (final concentration is 0.1 mM) is added, and induced expression is carried out at 220 rpm at 16 ℃ for 16 to 20 hours.
(4) CPP-mCherry fusion protein purification and concentration: the bacterial liquid for inducing expression is centrifuged for 15 min at 6000 rpm and 4 ℃ to collect bacterial cells, 30mL of PBS solution is added into the bacterial cells, the bacterial cells are peeled off from the wall of a centrifugal bottle by a glass rod, and then the bacterial cells are thoroughly resuspended on a vortex instrument (the bacterial cells obtained by 500mL of bacterial liquid are dissolved in 30mL of PBS at most); the resuspended cells are crushed for 15 min by ultrasonic, the cells are subjected to ultrasonic treatment for 2s and are stopped for 4s, and the cells are operated in a dark environment in the ultrasonic treatment process. Transferring the crushed bacterial liquid into a 50 mL centrifuge tube, balancing, centrifuging at 4 ℃ and 10000 rpm for 30 min, and collecting a supernatant; after balancing the Co column by 3-5 column volumes through PBS, passing the collected supernatant through the column, and repeating the column hanging for one time; washing 3-5 column volumes with PBS for removing impurities such as cell debris which cannot be removed by high-speed centrifugation; washing 3-5 column volumes with PBS containing 5 mM imidazole for removing non-specifically bound hybrid proteins from the gel; washing 5-8 column volumes with PBS containing 150 mM imidazole to dissociate target proteins specifically bound to the gel, and collecting all eluates; washing 3-5 column volumes with PBS containing 500 mM imidazole, filling the collected proteins into dialysis bags, and immersing in PBS solution to displace imidazole in the collection solution; since the target protein is fluorescent, the dialysis device is placed at 4 ℃ overnight in a dark place; changing the dialysate once in the next day; placing the dialyzed protein into a protein concentration tube, centrifuging at 4 ℃ and 4000 g, and concentrating the protein to 1.5-2 mL with the concentration of about 150 mu M; the whole experimental operation is protected from light; the obtained protein was stored at-80℃in a freeze-protected manner.
(5) Cobalt affinity chromatography column renaturation: first using ddH 2 O washing 6-8 column volumes, then 2 column volumes with 0.4N NaOH; immediate use of ddH 2 O-washing 6-8 column volumes followed by 0.1M EDTA wash column with ddH 2 Immediately after O-washing 6-8 column volumes, the column was washed with 0.1M CoCl 2 Washing 5-8 column volumes and incubating for 15 min at room temperature; incubation was completed with a large amount of ddH 2 After O washing, 3-5 column volumes are balanced by PBS, and the product can be stored for a long time at 4 ℃.
Example 2
A foreign protein introduction method suitable for streptomycete living cells comprises the following steps:
(1) Treatment of Streptomyces spores: adding 4 mL PBS solution on a streptomycete flat plate, lightly scraping the streptomycete spores by an inoculating loop, blowing and uniformly mixing by a pipetting gun, uniformly dividing bacterial liquid into a centrifuge tube, centrifuging at 4000 rpm for 3 min, collecting bacterial cells, washing for 2 times by PBS, and sucking out redundant solution to obtain the streptomycete sporophytes.
(2) Taking the same thallus quantity, adding Tat-mCherry, ANTP-mCherry (KFF) respectively 3 K-mCherry、K 9 -mCherry、R 9 -mCherry、(KH) 9 BP100-mCherry was diluted uniformly to 50. Mu.M with PBS. Taking 100 mu L of the diluent, adding the diluent into cells, gently mixing the mixture, and incubating the mixture at 34 ℃ in a dark place for 1 h; 50. Mu.M fluorescent protein mCherry without a transmembrane peptide fragment was used as a control.
(3) After incubation, centrifuging at 10000 rpm for 1 min, removing the supernatant, adding 500 mu L of PBS, re-suspending and washing once, centrifuging, removing the supernatant, adding 100 mu L of 0.25% trypsin, and digesting at 37 ℃ for 3-5 min to remove proteins adsorbed on the surface of the thalli; after centrifugation to remove trypsin and washing twice with PBS, the cells were resuspended in 500. Mu.L of PBS.
(4) Fluorescence microscopy (all samples were inserted in ice protected from light before preparing the observation samples): the microscope was opened 20 min in advance for preheating, and after fluorescence stabilized, 5. Mu.L of the resuspended bacterial liquid was pipetted onto the slide, coverslipped, and fluorescence microscopy was performed (FIG. 2).
(5) The efficiency and fluorescence intensity of different membrane penetrating peptides for mediating the cell penetration of the fluorescent protein mCherry are counted, the optimal membrane penetrating peptide suitable for streptomycete in the tested range is selected, the condition of the membrane penetrating peptide for mediating the cell penetration of the fluorescent protein is optimized, and the influence of the membrane penetrating peptide on the activity of streptomycete cells is examined, and the results are shown in figures 3-7.
Claims (5)
1. The method for introducing exogenous proteins into the live cells of streptomycete is characterized by comprising the following steps of:
(1) Treatment of Streptomyces spores: adding PBS solution on a streptomycete flat plate, scraping streptomycete spores by an inoculating loop, blowing and uniformly mixing by a pipetting gun, uniformly dividing bacterial liquid into centrifuge tubes, centrifugally collecting bacterial bodies, washing for 2 times by using PBS, and sucking out redundant solution to obtain streptomycete sporophytes;
(2) Adding Streptomyces sporophytes into cell penetrating peptide-target protein complex, wherein the cell penetrating peptide-target protein complex is R 9 -mCherry or (KH) 9 -BP100-mCherry, diluted to 50 μm with PBS, 100 μl of the dilution was added to the cells, mixed well, incubated at 34 ℃ in the dark for 1 h;
(3) After incubation, centrifuging to remove the supernatant, adding PBS for resuspension and washing once, centrifuging to remove the supernatant, adding 0.25% trypsin, digesting for 3-5 min at 37 ℃, centrifuging, washing twice with PBS, and resuspension the thalli with PBS;
the preparation method of the cell penetrating peptide-target protein complex in the step (2) comprises the following steps:
2-1) construction of a CPP-mCherry protein expression plasmid pET28a-CPP-mCherry: constructing a membrane penetrating peptide, 4 glycine connecting peptides and fluorescent protein mCherry fusion expression gene in an escherichia coli expression plasmid pET-28a (+);
2-2) transformation of plasmid pET28a-CPP-mCherry into E.coli by Calcification transformationE.coliBL21 (DE 3) competent cells;
2-3) inoculating CPP-mCherry recombinant expression strain into 5mL LB liquid medium containing kanamycin, activating 8 h at 37 ℃ and 220 rpm, inoculating the activated strain into 250 mL 2 XYT medium with the volume ratio of 1:100, adding 250 mu L of 50-55 mg/mL kanamycin, and culturing 4.5-5.5 h until OD is reached 600 The value reaches 0.6-0.8, the bacterial liquid is cooled to 16 ℃, IPTG is added, and induced expression is carried out for 16-20 h at 16 ℃ and 220 rpm;
2-4) CPP-mCherry fusion protein purification and concentration: centrifuging the bacteria liquid for induced expression, collecting the bacteria, adding 30mL of PBS solution into the bacteria, peeling the bacteria, thoroughly re-suspending the bacteria on a vortex instrument, ultrasonically crushing the re-suspended bacteria, centrifuging the crushed bacteria liquid, and collecting the supernatant; after the cobalt affinity chromatography column is balanced for 3-5 column volumes by PBS, passing the collected supernatant through the column, and repeating column hanging for one time; washing 3-5 column volumes with PBS, washing 3-5 column volumes with PBS containing 5 mM imidazole, washing 5-8 column volumes with PBS containing 150 mM imidazole, and collecting all eluates; washing 3-5 column volumes with PBS containing 500 mM imidazole, dialyzing the collected proteins, and concentrating to obtain cell penetrating peptide-target protein complex;
wherein, the penetrating peptide and the amino acid sequence thereof in the step 2-1) are one of the following:
R 9 ：RRRRRRRRR；
KH 9 -BP100：KHKHKHKHKHKHKHKHKHKKLFKKILKYL；
the cell penetrating peptide-target protein complex obtained correspondingly in the step 2-4) is R 9 -mCherry or (KH) 9 -BP100-mCherry。
2. The method for introducing exogenous proteins into a living cell of Streptomyces according to claim 1, wherein the method comprises the steps of: in the step 2-3), the kanamycin concentration in the LB liquid medium is 50-55 mug/mL, and the IPTG final concentration is 0.1-0.4 mM.
3. The method for introducing exogenous proteins into a living cell of Streptomyces according to claim 1, wherein the method comprises the steps of: in the step 2-4), the ultrasonic breaking time of the re-suspended thalli is 15 min, the ultrasonic is carried out for 2s, the stop is carried out for 4s, and the ultrasonic process is operated in a dark environment.
4. The method for introducing exogenous proteins into a living cell of Streptomyces according to claim 1, wherein the method comprises the steps of: in the step 2-4), the specific steps of protein dialysis and concentration are as follows: and (3) filling the collected proteins into a dialysis bag, immersing the dialysis bag in PBS solution, dialyzing overnight at 4 ℃ in the dark, placing the dialyzed proteins into a protein concentration tube, centrifuging, concentrating the proteins to 150 mu M, and performing experiment operation in the dark.
5. The method for introducing exogenous proteins into a living cell of Streptomyces according to claim 1, wherein the method comprises the steps of: also comprises the steps of 2-5), cobalt affinity chromatography column renaturation: with ddH 2 O-washing 6-8 column volumes, followed by 2 column volumes with 0.4N NaOH, immediately with ddH 2 O-washing 6-8 column volumes followed by 0.1M EDTA wash column with ddH 2 Immediately after O-washing 6-8 column volumes, the column was washed with 0.1M CoCl 2 Washing 5-8 column volumes and incubating at room temperature for 15 min, with a large amount of ddH at the completion of incubation 2 After the O-wash, 3-5 column volumes were equilibrated with PBS.
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