CN106350494B

CN106350494B - A method for anchoring and optimizing methyl parathion hydrolase with flavin fluorescent protein

Info

Publication number: CN106350494B
Application number: CN201610753732.7A
Authority: CN
Inventors: 张贞; 马立新; 卞璐; 唐荣兴; 沈威
Original assignee: Hubei University
Current assignee: Hubei University
Priority date: 2016-08-29
Filing date: 2016-08-29
Publication date: 2020-05-19
Anticipated expiration: 2036-08-29
Also published as: CN106350494A

Abstract

The present invention proposes a method for anchoring and optimizing the display of methyl parathion hydrolase by using flavin fluorescent protein. The steps are: 1) construct fusion gene (H ₆ MPH-EcFbFP); 2) construct recombinant plasmid pET23a/H ₆ MPH-EcFbFP, and simultaneously introduce the coding sequence of charged polypeptide 6×Glu into fusion protein (H ₆ ) by PCR MPH-EcFbFP) coding gene 3' end, and construct recombinant plasmid pET23a/H ₆ MPH-EcFbFP _(E6) ; 3) recombinant plasmid transforms Escherichia coli competent cells to obtain genetically engineered strains; 4) recombinant strains are cultured in shake flasks , and extract each component of the cell respectively to detect the surface display efficiency; 5) measure the enzymatic activity of methyl parathion hydrolase; 6) measure the stability of MPH surface display. The invention uses flavin fluorescent protein (EcFbFP) as an anchor protein for the first time to display MPH on the surface of Escherichia coli, with high display efficiency and good stability of the prepared MPH enzyme activity.

Description

Method for anchoring and optimally preparing methyl parathion hydrolase by using flavin fluorescent protein

Technical Field

The invention relates to a surface technology of Methyl Parathion Hydrolase (MPH), in particular to a method for improving the display efficiency of the Methyl Parathion Hydrolase (MPH) by optimizing charges carried by a novel anchoring protein, namely flavin fluorescent protein (EcFbFP), and belongs to a novel idea, a novel way and a novel method for displaying the Methyl Parathion Hydrolase (MPH) on the surface of escherichia coli.

Background

Around 40 million tons of pesticide are consumed for agricultural control every year worldwide, and the organophosphorus pesticide accounts for 70% of the total pesticide usage, but the effective utilization rate is less than 1%. With the long-term and large-scale use of organophosphorus pesticides, more and more environmental problems, human health problems and sustainable development problems are increasingly highlighted. Therefore, the method for biologically degrading organophosphorus pesticide safely, economically and effectively is urgently sought. The method for degrading organophosphorus pesticides by microbial enzyme systems is paid more attention by people on the basis of low cost and high activity.

The rapid development of the research on the surface display system of heterologous protein and the anchoring strategy thereof provides a new strategy for the production of high-activity and low-cost organophosphorus hydrolase, and solves the problems that the degrading enzyme produced by the traditional recombinant engineering strain cannot freely pass through a cell membrane structure, so that the activity of the enzyme cannot be fully exerted in the application process, the degrading enzyme is separated and purified from the engineering strain, the popularization and the application of the degrading enzyme are limited due to complicated process and high cost, and the like.

Methyl Parathion Hydrolase (MPH) is one of the members in the organophosphorus hydrolase family, is derived from Pseudomonas sp, mainly degrades methyl parathion and also degrades ethyl parathion, and exists in organophosphorus pesticides such as fenitrothion and chlorpyrifos. At present, a series of achievements are obtained by researching the cell surface display technology of Methyl Parathion Hydrolase (MPH) at home and abroad, and Escherichia coli MPH display engineering bacteria are successfully constructed. For example, Yang et al^[3]Fusing a signal peptide TorA of a Tat signal channel to the amino terminal of MPH to realize the expression of the polypeptide in the periplasm of the cells, and measuring the periplasm of the cellsThe enzyme activity in the cytoplasm is 3 times that of cytoplasm expression; yang et al^[2]Anchoring protein Lpp-OmpA; AIDA and the like are respectively fused with the amino terminal of the MPH to realize the display of the MPH on the cell surface of escherichia coli, wherein the enzyme activity of the MPH displayed by Lpp-OmpA as an anchoring protein reaches 1.52U/OD₆₀₀。

However, the existing MPH display system using the Escherichia coli surface display system has some defects, such as: (1) errors often occur in the fixed part, so that MPH expression is unstable; (2) the expressed MPH has low activity and short activity retention time; (3) MPH is toxic to Escherichia coli, and causes growth arrest and autolysis of cells in the expression process. These directly affect the expression of MPH and the efficiency of degrading organophosphorus pesticides.

Reference to the literature

[1]Shhimazu M,Mulchandani A,Chen W.Cell surface display oforganophosphorus hydrolase using ice nucleation protein[J].Biotechnol Prog,2001；17(1):76-80.

[2]Yang JJ,Liu RH,Jiang H,Yang Y,Qiao CL.Selection of a whole-cellbiocatalyst for methyl parathion biodegradation[J].Appl Microbiol Biotechnol,2012；95:1625-1632.

[3]Yang C,Freudl R,Qiao CL.Export of methyl parathion hydrolase tothe periplasm by the twin-arginine translocation pathway in Escherichia coli[J].J Argric Food Chem,2009,57:8901-8905.

Disclosure of Invention

The invention aims to provide a method for anchoring and optimally preparing methyl parathion hydrolase by using flavin fluorescent protein, which improves the surface display efficiency of the Methyl Parathion Hydrolase (MPH) in escherichia coli and improves the stability of the prepared Methyl Parathion Hydrolase (MPH).

The present invention is thus achieved. The method comprises the steps of constructing a fusion protein (H6MPH-EcFbFP) by using a flavin fluorescent protein (EcFbFP) as an anchor protein, and improving the display efficiency of the fusion protein (H6MPH-EcFbFP) on the cell surface of escherichia coli by optimizing the charge of the flavin fluorescent protein (EcFbFP);

1) and (5) constructing a recombinant plasmid. Firstly, designing and constructing a fusion gene (H6 MPH-EcFbFP); secondly, cloning the fusion gene (H6MPH-EcFbFP) to an expression vector pET23a-T (constructed by the laboratory or purchased externally), and constructing a recombinant expression plasmid pET23a/H6 MPH-EcFbFP;

then, designing a PCR primer to carry a gene sequence coding the polypeptide 6 XGlu with negative charge, introducing a recombinant plasmid pET23a/H6MPH-EcFbFP by PCR, and reconstructing a recombinant expression plasmid pET23a/H6MPH-EcFbFP_(E6)(the structure schematic diagram is shown in figure 1 and figure 2, the amino acid sequence is shown in table 1, and the PCR primer is shown in table 2);

TABLE 1 Signal peptides and charged polypeptides and their amino acid sequences

TABLE 2 primer names and primer sequences

The direction of the primer is 5 '-3'

2) And (5) constructing a recombinant strain. Transforming the two constructed recombinant plasmids into an escherichia coli expression strain Rosetta Blue competent cell, coating the Escherichia coli expression strain Rosetta Blue competent cell on a LA plate (the final concentration of ampicillin is 100 mu g/mL), and standing and culturing at 37 ℃ overnight to obtain a recombinant strain;

3) culturing and expressing the recombinant strain. Inoculating the genetic engineering strain into 100mL LB culture medium, wherein the final concentration of ampicillin is 100 mug/mL, shake-culturing at 37 ℃ on a shake flask with 200RPM, the OD value is 0.5-0.6, adding IPTG (isopropyl-beta-thiogalactoside) with the final concentration of 1mM, and carrying out induction culture at 37 ℃ for 8 hours. Then, the thalli is centrifugally collected at 12000RPM and 4 ℃ for 10 minutes, washed for 3 times by precooled phosphate buffer and resuspended in 10mL of precooled phosphate buffer for later use;

4) extracting each component of the cell. Collecting 1mL of bacterial solution, centrifuging at 12000RPM and 4 deg.C for 2 min, collecting thallus, and resuspending the thallus in 1Standing in mL precooled TES buffer solution for 5 minutes at 12000RPM, and centrifuging for 10 minutes, wherein the centrifuged supernatant is the cell envelope component; resuspend pellet in precooled MgCl₂Standing in a buffer solution at 4 ℃ for 30 minutes at 12000RPM, centrifuging for 10 minutes, and obtaining a supernatant after centrifugation as a periplasm component; the pellet was resuspended in pre-chilled PBS buffer to obtain the cytosolic fraction.

5) 200. mu.L of each of the above cell fractions in step 4 was collected and examined by SDS-PAGE. After Coomassie brilliant blue staining, calculating the proportion of target protein in each component in the total target protein by adopting a gray scanning method so as to calculate the display efficiency of the fusion protein;

6) whole cell MPH activity assay of the strain. According to the literature^[1]The method of (1) performs activity measurement on the whole-cell MPH of the strain.

The IPTG-induced cell cultures were harvested, washed 3 times with citrate-phosphate buffer at pH 8.0, resuspended in 50. mu.M CoCl₂In a citric acid-phosphate buffer solution, and adjusting the OD thereof₆₀₀Is 1.0. The enzyme activity reaction system (1000 mu L solution) comprises: OD₆₀₀A200. mu.L suspension of 1.0 was reacted at 37 ℃ for 2 minutes, and the change in absorbance at 410nm was measured. 1 MPH enzyme activity unit (U) is defined as the amount of enzyme required to hydrolyze 1 μ M paraoxon per minute;

7) and (3) performing displayed stability detection, namely performing enzyme activity determination on the same sample in the same time every day by referring to the method in the step 6, continuously measuring for 31 days, and drawing the change trend of the enzyme activity.

The invention relates to a display method for displaying methyl parathion hydrolase on the cell surface of escherichia coli, which comprises a display method for displaying flavofluorescence protein (EcFbFP) as an anchor protein and Methyl Parathion Hydrolase (MPH) as a displayed target protein, and a display method for displaying Methyl Parathion Hydrolase (MPH) by the flavofluorescence protein (EcFbFP) optimized on the basis.

The invention has the advantages.

Firstly, the invention establishes a brand-new escherichia coli cell surface display system taking flavin fluorescent protein (EcFbFP) as anchoring protein; secondly, benefit fromBy using the display system, the Methyl Parathion Hydrolase (MPH) realizes the display on the surface of an escherichia coli cell, the display efficiency of the methyl parathion hydrolase on the surface of the escherichia coli is improved by about 5 times and the display enzyme activity is improved by about 10 times by optimizing the charge carried by flavin fluorescent protein; with Yang et al^[2]Reported methyl parathion hydrolase activity (1.52U/OD) displayed using (LPP-OmpA) as dockerin₆₀₀) (ii) proximity of (a); after 31-day continuous measurement, the enzyme activity trend of the recombinant strain is analyzed, and the MPH displayed by the system still has 100 percent of residual enzyme activity on the 14 th day, and the recombinant strain still has nearly 50 percent of residual enzyme activity on the 31 th day, and the activity of the recombinant strain is similar to that of Yang and the like^[2]Compared with the reported LPP-OmpA as the anchoring protein displayed methyl parathion hydrolase display stability which is sharply reduced (the residual enzyme activity is reduced to less than 30 percent on day 3), the display system has good heterologous protein display stability.

Drawings

Fig. 1 and 2 show experimental design schemes of the present invention. As shown, EcFbFP is the english abbreviation for flavin fluorescent protein (EcFbFP); MPH is English abbreviation of methyl parathion hydrolase; his is the English abbreviation of tag protein; e6 is an english abbreviation for six glutamic acids. The design scheme is as follows, firstly, construct the recombinant plasmid pET23a/H₆MPH-EcFbFP, and then introducing the charged polypeptide (6 XGlu) into the carboxyl terminal of the flavin fluorescent protein (EcFbFP) to construct a recombinant plasmid pET23a/H₆MPH-EcFbFP_(E6)。

FIG. 3 shows the efficiency of SDS-PAGE analysis and determination of the MPH enzyme activity on the surface. 1 is a fusion protein H₆The analysis result of MPH-EcFbFP in each component of the cell shows that C is the total protein of the cytoplasmic component of the cell; p is the total protein of the periplasmic component of the cell; OM is the component total protein of the cell outer membrane. 2 is a fusion protein H₆MPH-EcFbFP_(E6)The result of analysis in each fraction of the cell, C is the total protein of the cytoplasmic fraction of the cell; p is the total protein of the periplasmic component of the cell; OM is the component total protein of the cell outer membrane.

FIG. 4 shows the results of measuring the enzyme activity of surface-displayed MPH, 1 shows the measurement of surface-displayed fusion protein H₆The enzyme activity result of MPH-EcFbFP;2 for determination of surface display fusion protein H₆MPH-EcFbFP_(E6)The enzyme activity of (2) is obtained.

FIG. 5 shows the stability of organophosphorous hydrolase on the surface of the analyzed cells. In the figure is the surface display fusion protein H₆A trend graph of the stability determination results of MPH-EcFbFP;

FIG. 6 shows the stability of organophosphorous hydrolase on the surface of the analyzed cells. In the figure is the surface display fusion protein H₆MPH-EcFbFP_(E6)Trend graph of stability assay results.

Detailed Description

The invention is further illustrated by the following examples:

example 1:

the method of the invention is utilized to display the Methyl Parathion Hydrolase (MPH) on the cell surface of the escherichia coli. Firstly, the constructed recombinant plasmid pET23a/H₆MPH-EcFbFP is transformed into an Escherichia coli competent cell Rosetta Blue strain, and the strain is statically cultured at 37 ℃ overnight. Then, a single colony was picked and inoculated in 100mL of LB medium, the final concentration of the antibiotic ampicillin was 100. mu.g/mL, shake-cultured at 37 ℃ with OD value of 0.5-0.6, IPTG was added to the medium at 1mM, and induction-cultured at 37 ℃ for 8 hours. Then, the cells were collected by centrifugation at 12000RPM at 4 ℃ and washed 3 times with pre-chilled PBS, and finally resuspended in 10mL of pre-chilled PBS buffer for further use. Analysis was then performed by reference to the methods in step 4, step 5 of the summary of the invention, and enzyme activity measurements were performed by reference to the methods in step 6 of the summary of the invention (FIG. 3).

Example 2:

the system for the cell surface display of methylparathion hydrolase (MPH) in E.coli was optimized. Firstly, on the basis of recombinant expression plasmid pET23a/H6MPH-EcFbFP, designing PCR primer to carry gene sequence coding polypeptide 6 XGlu with negative charge, introducing the gene sequence into recombinant plasmid pET23a/H6MPH-EcFbFP by PCR to construct recombinant expression plasmid pET23a/H6MPH-EcFbFP_(E6)(ii) a Secondly, the constructed recombinant plasmid pET23a/H₆MPH-EcFbFP_(E6)Escherichia coli competent cell Rosetta Blue strain was transformed, and cultured overnight at 37 ℃. Then, a single colony was picked up and inoculated into 100mL of LB medium,the final concentration of the antibiotic ampicillin is 100 mug/mL, shaking culture is carried out at 37 ℃, the OD value is 0.5-0.6, IPTG is added, the final concentration is 1mM, and induction culture is carried out for 8 hours at 37 ℃. Then, the thalli are collected at 12000RPM and 4 ℃ in a centrifugal mode, washed for 3 times by precooled PBS, and finally resuspended in 10mL of precooled PBS buffer solution for later use; then, analysis was performed by referring to the method in step 4 and step 5 of the summary of the invention, and enzyme activity measurement was performed by referring to the method in step 6 of the summary of the invention (FIG. 4).

Example 3:

the cells collected by centrifugation in examples 1 and 2 were washed 3 times with pre-cooled PBS and finally resuspended in 10mL of pre-cooled PBS buffer for further use. Then, referring to the summary of the invention, the method in step 7) was performed on the same broth at the same time point every day for enzyme activity measurement (fig. 5, fig. 6).

Analysis of various example results

The flavin fluorescent protein (EcFbFP) is used as an anchor protein to realize the display of the Methyl Parathion Hydrolase (MPH) on the cell surface of the escherichia coli for the first time, and the displayed methyl parathion hydrolase has activity. After the charge of the flavin fluorescent protein (EcFbFP) is optimized, the display efficiency is improved to 50 percent from the original 10 percent, and the enzyme activity of the displayed methyl parathion hydrolase is changed from 0.17 +/-0.002U/OD₆₀₀The yield is improved to 1.099 +/-0.005U/OD₆₀₀The enzyme activity is improved by nearly 10 times, and Yang and the like^[2]Reported methyl parathion hydrolase activity (1.52U/OD) displayed using LPP-OmpA as dockerin₆₀₀) Is close to each other. The invention also measures the display stability of the methyl parathion hydrolase displayed by the system, and through continuous measurement for 31 days, the enzyme activity trend of the methyl parathion hydrolase is analyzed to find that the MPH displayed by the system still has 100 percent of residual enzyme activity on the 14 th day, and the recombinant strain still has nearly 50 percent of residual enzyme activity on the 31 th day, which is similar to Yang and the like^[2]Compared with the reported LPP-OmpA as the anchoring protein displayed methyl parathion hydrolase display stability which is sharply reduced (the residual enzyme activity is reduced to less than 30 percent on day 3), the display system has good heterologous protein display stability.

The Escherichia coli strain Rosetta Blue is purchased from Novagen, USA, different recombinant plasmids are constructed in the laboratory or purchased externally, and various reagents for analysis are analytical reagents.

Claims

1. one utilizes flavin fluorescent protein to anchor and optimize the method for displaying methyl parathion hydrolase, it is characterized in that step is:

1) Construction of recombinant plasmid: first, design and construct fusion gene H ₆ MPH-EcFbFP; secondly, clone fusion gene H ₆ MPH-EcFbFP into expression vector pET23a-T, and construct recombinant expression plasmid pET23a/H ₆ MPH-EcFbFP; then, The PCR primers were designed to carry the gene sequence encoding the negatively charged polypeptide 6×Glu, and the gene sequence encoding 6×Glu was introduced into the recombinant plasmid pET23a/H ₆ MPH-EcFbFP by PCR to construct the recombinant expression plasmid pET23a/H ₆ MPH-EcFbFP _(E6) , to introduce the negatively charged polypeptide 6×Glu into the carboxyl terminus of the flavin fluorescent protein EcFbFP; the amino acid sequence of the flavin fluorescent protein EcFbFP is shown in the sequence table SEQID NO.1; the amino acid sequence of methyl parathion hydrolase MPH is shown in the sequence The list is shown in SEQ ID NO.2;

2) Construction of recombinant strains: The two constructed recombinant plasmids were transformed into E. coli expression strain Rosetta Blue competent cells, spread on LA plates, and cultured at 37°C for 16 hours to obtain recombinant strains;

3) Recombinant strain culture and expression: Inoculate the recombinant strain in LB medium with an ampicillin concentration of 100 μg/mL for induction and culture, and the culture conditions are: first, 37 ° C 200 RPM shaking culture, at an OD value between 0.5 and 0.6, add IPTG , the final concentration was 1 mM, and the induction culture was continued for 8 hours at 37 °C; then, the cells were collected by centrifugation at 12,000 RPM, 4 °C, 10 minutes, and the cells were washed 3 times with pre-cooled PBS and resuspended in 10 mL of pre-PBS for use;

4) Extraction of cell components: take 1 mL of bacterial solution, centrifuge at 12000RPM, 4°C for 2 minutes to collect bacteria, resuspend the bacteria in 1mL of pre-cooled TES buffer, stand for 5 minutes, centrifuge at 12000RPM for 10 minutes, and centrifuge The supernatant was defined as the extracellular membrane fraction; the pellet was resuspended in 1 mL of pre-cooled MgCl ₂ buffer, let stand at 4°C for 30 minutes, 12000 RPM, and centrifuged for 10 minutes, and the supernatant after centrifugation was defined as the periplasm Component; resuspend the pellet in 1 mL of pre-chilled PBS buffer, defined as the cytoplasmic component;

5) Determination of cell surface display efficiency: Take 200 μL of the above-mentioned cell components in step 4 and detect them by SDS-PAGE; after staining with Coomassie brilliant blue, use grayscale scanning to calculate the proportion of the target protein in each component. The ratio of the total target protein to calculate the display efficiency of MPH;

6) Cell surface display MPH activity assay:

Cell cultures induced by IPTG were collected, washed 3 times with citric acid-phosphate buffer pH to 8.0, resuspended in citric acid _- phosphate buffer containing 50 μM CoCl, and adjusted to an OD ₆₀₀ of 1.0, 1000μL enzyme activity reaction system contains 200μL of bacterial suspension with OD ₆₀₀ of 1.0, react at 37°C for 2 minutes, measure the change of absorbance at 410nm, 1 MPH enzyme activity unit (U) is defined as hydrolysis of 1μM formazan per minute. The amount of enzyme required for base paraoxon;

7) Demonstrate MPH stability assay: referring to the method in step 6, the same sample was tested for enzyme activity at the same time every day for 31 consecutive days, and the change trend of enzyme activity was drawn.