CN117487841B - Method for constructing difunctional yeast display and secretion system by using 2A peptide strategy - Google Patents

Abstract

The invention discloses a method for constructing a difunctional yeast display and secretion system by using a 2A peptide strategy, which relates to the technical field of constructing difunctional yeast by using a 2A peptide strategy, and constructs a recombinant plasmid pYD1-F2A-GFP by taking green fluorescent protein as a target protein, inserting a 2A peptide sequence from foot-and-mouth disease virus into an Aga2 expression cassette of a pYD1 plasmid and connecting the sequence with a downstream target gene GFP; the recombinant plasmid is transformed into the yeast strain EBY100, and through the steps of positive clone screening, target protein induced expression, extraction and identification, the problem that the target protein can only be displayed on the cell surface and cannot be secreted into the culture medium supernatant at the same time in the traditional yeast display system is solved.

Description

Method for constructing difunctional yeast display and secretion system by using 2A peptide strategy

Technical Field

The invention belongs to the technical field of construction of difunctional yeast by using a 2A peptide strategy, and particularly relates to a method for constructing a difunctional yeast display and secretion system by using a 2A peptide strategy.

Background

The 2A peptide, also known as a "self-cleaving" peptide, is an oligopeptide, typically 18-22 amino acids, located between two proteins of some members of the picornavirus family. The 2A peptide functions by allowing the ribosome to skip synthesis of glycine (Gly) and proline (Pro) peptide bonds at the C-terminus of the 2A element. Based on this feature, 2A peptide is often used for the isolation of multiple translation products, i.e., a DNA fragment encoding the 2A peptide can be inserted between the coding regions of two proteins, such that the proteins undergo self-cleavage after translation is completed, separating into two independently folded proteins. The method can avoid the use of conventional protein separation methods such as column chromatography, electrophoresis, etc., thereby avoiding damage and contamination to proteins. However, the self-cleavage efficiency of the 2A peptide is affected by the upstream and downstream open reading frame sequences, and therefore, a fusion protein that is not able to effectively self-cleave would be another translation product.

The yeast surface display system is a eukaryotic cell expression system that can display foreign proteins on the yeast surface. In this system, the gene sequence of the target protein is fused with a specific vector gene sequence and then introduced into yeast cells by chemical transformation or the like. The protein of interest can be produced under specific medium induction and can be transferred to the cell surface and immobilized on the cell wall by the intracellular protein transport mechanism of yeast. Unlike phage surface display, yeast cells in the yeast surface display system have a molecular chaperone system with more complete functions, which can assist in folding and modification of proteins, thereby affecting the structure and function of the proteins. Furthermore, unlike phage, yeast cells are large enough particles to be sorted by flow cytometry, which makes possible the isolation and selection of mutants based on specific quantitative affinity changes. In addition to screening and engineering functional polypeptides, yeast surface display technology can also be used in bioadsorption, biofermentation, biosynthesis, and the like. However, the conventional yeast display technology can only display the target protein on the cell surface, but cannot secrete the target protein into the supernatant of the culture medium, and subsequent steps of target protein extraction, purification and the like not only increase the difficulty of obtaining the full-length target protein, but also increase the cost and period of experiments. Therefore, the construction of the difunctional yeast display and secretion system which can display the target protein on the cell surface and secrete the target protein into the supernatant of the culture medium has a certain practical application value.

Disclosure of Invention

The invention aims to solve the problem that the target protein in the traditional yeast display system can only be displayed on the cell surface and cannot be secreted into the culture medium supernatant at the same time. Therefore, the invention provides a method for constructing a bifunctional yeast display and secretion system by applying a 2A peptide strategy.

A method for constructing a bifunctional yeast display and secretion system by using a 2A peptide strategy, which specifically comprises the following steps:

inserting a 2A peptide sequence derived from foot-and-mouth disease virus into an Aga2 expression cassette of a pYD1 plasmid by taking green fluorescent protein as a target protein and connecting the sequence with a downstream target gene GFP to construct a recombinant plasmid pYD1-F2A-GFP;

the recombinant plasmid is transformed into a yeast strain EBY100, and through the steps of positive clone screening, target protein induced expression, extraction and identification, a novel yeast display system with the functions of protein secretion and surface display is constructed by utilizing the characteristic that the 2A peptide is sheared by itself but cannot be sheared effectively each time.

Compared with the prior art, the invention has the beneficial effects that:

the method solves the problem that the target protein in the traditional yeast display system can only be displayed on the cell surface and cannot be secreted into the culture medium supernatant at the same time.

Drawings

FIG. 1 is a schematic diagram of recombinant plasmid pYD1-F2A-GFP;

FIG. 2 is a PCR identification of transformants of the present invention;

FIG. 3A is a schematic diagram showing the results of the target protein in the detection of the exoprotein of the present invention;

FIG. 3B is a schematic diagram showing the results of the target protein in the detection of the display protein according to the present invention.

Detailed Description

The technical solutions of the present invention will be clearly and completely described in connection with the embodiments, and it is obvious that the described embodiments are only some embodiments of the present invention, not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Referring to FIG. 1, the present application provides a method for constructing a bifunctional yeast display and secretion system using a 2A peptide strategy,

as an embodiment of the present invention, the method specifically includes the steps of: the Green Fluorescent Protein (GFP) is taken as a target protein, a 2A peptide (F2A) sequence from foot-and-mouth disease virus is inserted into an Aga2 expression cassette of a pYD1 plasmid and connected with a sequence of a downstream target gene GFP, so as to construct a recombinant plasmid pYD1-F2A-GFP.

The recombinant plasmid is transformed into a yeast strain EBY100, and through the steps of positive clone screening, target protein induced expression, extraction, identification and the like, a novel yeast display system with dual functions of protein secretion and surface display is constructed by utilizing the characteristic that 2A peptide is sheared by itself but cannot be sheared effectively each time.

As a further embodiment of the present invention, the method provided by the present invention specifically includes:

construction of recombinant plasmids

Recombinant, granulated and transformed Saccharomyces cerevisiae

Positive clone screening

Induction expression and extraction of target protein

Identifying a target protein Western Blot;

the construction method of the specific recombinant plasmid comprises the following steps:

1) Linearization of vector plasmid: and (3) adopting NheI and MluI to enzyme-cut the pYD1 vector, and recovering the glue for later use. The 50. Mu.l cleavage system was as follows: 5 μg of pYD1 vector; 10 XrCutSmartBuffer 5. Mu.l; nheI1 μl; mluI 1. Mu.l; sterile water was made up to 50 μl. The above reagents were mixed and placed in a 37℃water bath for 1h.

2) Homologous recombination of target fragment: the target fragment comprises a Flag tag sequence, a foot-and-mouth disease virus 2A peptide sequence and a GFP sequence, gene synthesis is carried out by the Kirschner biotechnology, the target fragment is mixed with a linearization carrier, homologous recombination reaction is carried out under the catalysis of homologous recombinase, and a recombinant plasmid pYD1-F2A-GFP is constructed, as shown in figure 1. The 20. Mu.l homologous recombination system is as follows: 3 μl of the target fragment; linearizing 2 μl of plasmid; 2 μl of ExnaseII; 5 XCEBuffer 4. Mu.l; sterile water 9 μl. The above reagents were mixed and placed in a 37℃water bath for 2h.

The specific sequence of the recombinant plasmid is shown in SEQ ID NO.1 in the sequence table;

wherein, further, the recombinant graining transformed Saccharomyces cerevisiae comprises the following specific modes:

1) Taking one saccharomyces cerevisiae EBY100 conversion competence, and thawing on ice for standby;

2) 240ul of 50% PEG3350 was added;

3) 36ul of 1M LiAc was added;

4) 5ul ssDNA (20 mg/ml) was added;

5) 5ul of recombinant plasmid pYD1-F2A-GFP (100 ng/ul) was added;

6) Shaking vigorously for 1min, and completely mixing the above reagents;

7) Incubating in a water bath at 30 ℃ for 30min;

8) Heat shock in a water bath at a temperature of 42 ℃ for 25min;

9) Resuscitating in water bath at 30deg.C for 30min;

10 Centrifuging at 4000rpm for 5min, and discarding the supernatant;

11 Adding 200ul of sterile water to resuspend the thalli, gently mixing, and coating on an SD-Trp screening plate;

12 Culturing at 30℃for 4d.

Further, the positive clone screening and extraction concretely comprises the following steps:

8 clones were randomly selected from the plate and subjected to colony PCR.

The upstream primer sequence is as follows: see SEQ ID NO.2 of the sequence table;

the downstream primer sequences were: see SEQ ID NO.3 of the sequence Listing;

50. mu.l PCR system was as follows: DNA: 1. mu.l; 1 μl of the upstream primer; 1 μl of the downstream primer; 2X rapid Taq Master Mix. Mu.l; sterile water 22 μl.

The reaction procedure was as follows: 3min at 95 ℃;95 ℃ 15s; 15. 15s at 50 ℃;72 ℃ for 1min; at 72℃for 5min, 35 cycles were performed.

After the completion of PCR, 2. Mu.l of each sample was collected and subjected to agarose electrophoresis, and the result showed that the size of the target gene band was about 1170. 1170 bp, which was in accordance with the expectation, as shown in FIG. 2.

Further, the specific mode of the induction expression of the target protein is as follows:

1) Collecting all positive clones without mutation, diluting, inoculating into YNB-CAA liquid culture medium containing 2% glucose, shaking culturing at 30deg.C and 225rpm until OD600 = 2-5;

2) Centrifuging at 4000rpm for 5min, and discarding supernatant;

3) Adding YNB-CAA culture medium containing 2% galactose, blowing and mixing, adjusting the culture broth to OD600 = 0.5-1, 30 ℃, culturing at 225rpm, and shaking to induce the expression of target protein;

4) Collecting supernatant and surface displayed proteins, numbered 1-8 for secretion and 1-8 for display.

Further, the specific mode for identifying the target protein WesternBlot is as follows:

1) Adding appropriate amount of protein into 20 μl protein solution, and boiling at 100deg.C for 5-10min;

2) Centrifuging at 12000rpm for 2min, sucking 20 μl, and performing protein electrophoresis;

3) Cutting PVDF film with proper size, and transferring protein onto the film via wet film transferring process;

4) Washing the membrane for 10min by1 XTBST;

5) Taking a proper amount of sealing liquid to seal the membrane for 10min at room temperature;

6) Washing the membrane for 5min by1 XTBST;

7) The murine anti-GFP is selected as a primary antibody, and incubated for 2 hours at room temperature after dilution;

8) Washing the membrane for 10min by1 XTBST for 3 times;

9) HRP-labeled goat anti-mouse IgG is selected as a secondary antibody, and the secondary antibody is incubated for 45min at room temperature after dilution;

10 1 XTBE for 10min, total 3 times;

11 Taking ECL luminous liquid A, B, uniformly mixing the ECL luminous liquid A, B in equal quantity, dripping the ECL luminous liquid A, B on the front surface of the membrane, and exposing the ECL luminous liquid after incubation;

12 As shown in fig. 3A, the size of the target protein in the detection of the exoprotein is about 35KDa, including the target protein and the tag protein; as shown in fig. 3B, the size of the target protein in the detection of the display protein is about 45KDa, including the anchor protein, the F2A peptide, the target protein and the tag protein; at the same time, a protein was also detected at 35kDa in this figure, and this protein was substantially the same size as the secreted protein, probably because the protein being secreted, but not completely secreted, was extracted at the same time as the cell wall protein was extracted. The WesternBlot result shows that the yeast cells successfully secrete and display GFP.

The present application focuses on methods for constructing and validating systems for bifunctional yeast display and secretion using the 2A peptide strategy.

The above embodiments are only for illustrating the technical method of the present invention and not for limiting the same, and it should be understood by those skilled in the art that the technical method of the present invention may be modified or substituted without departing from the spirit and scope of the technical method of the present invention.

Claims (5)

1. A method for constructing a bifunctional yeast display and secretion system by using a 2A peptide strategy, which is characterized by comprising the following steps:

the green fluorescent protein is taken as a target protein, an F2A peptide sequence derived from foot-and-mouth disease virus is inserted into an Aga2 expression cassette of a pYD1 plasmid and connected with a sequence of a downstream target gene GFP, and a recombinant plasmid pYD1-F2A-GFP is constructed, and is shown in the following figure:

；

the recombinant plasmid is transformed into a yeast strain EBY100, and through the steps of positive clone screening, target protein induced expression, extraction and identification, a novel yeast display system with dual functions of protein secretion and surface display is constructed by utilizing the characteristic that F2A peptide is sheared by itself but cannot be sheared effectively each time;

the specific mode of constructing the recombinant plasmid is as follows:

1) Linearization of vector plasmid: cutting the pYD1 carrier by NheI and MluI enzyme, and recycling glue for later use; the 50. Mu.l cleavage system was as follows: 5 μg of pYD1 vector; 10 XrCutSmartBuffer 5. Mu.l; nheI1 μl; mluI 1. Mu.l; sterile water was made up to 50 μl;

mixing the reagents, and then placing the mixture in a water bath at 37 ℃ for 1h;

2) Homologous recombination of target fragment: mixing a target fragment with a linearization vector, and carrying out homologous recombination reaction under the catalysis of homologous recombination enzyme to construct a recombinant plasmid pYD1-F2A-GFP; the 20. Mu.l homologous recombination system is as follows: 3 μl of the target fragment; linearizing 2 μl of plasmid; 2 μl of ExnaseII; 5 XCEBuffer 4. Mu.l; sterile water 9 μl; mixing the above reagents, and placing in a water bath at 37 ℃ for 2 hours;

the target fragment comprises Flag tag sequence, F2A peptide sequence of foot-and-mouth disease virus and GFP sequence.

2. The method for constructing a bifunctional yeast display and secretion system using a 2A peptide strategy of claim 1, wherein the recombinant plasmid is transformed into Saccharomyces cerevisiae in the following specific manner:

1) Taking one saccharomyces cerevisiae EBY100 conversion competence, and thawing on ice for standby;

2) 240ul of 50% PEG3350 was added;

3) 36ul of 1MLiAc was added;

4) 5ul of ssDNA was added at a concentration of 20mg/ml;

5) 5ul of recombinant plasmid pYD1-F2A-GFP was added at a concentration of 100ng/ul;

6) Shaking vigorously for 1min, and completely mixing the above reagents;

7) Incubating in a water bath at 30 ℃ for 30min;

8) Heat shock in a water bath at a temperature of 42 ℃ for 25min;

9) Resuscitating in water bath at 30deg.C for 30min;

10 Centrifuging at 4000rpm for 5min, and discarding the supernatant;

11 Adding 200ul of sterile water to resuspend the thalli, gently mixing, and coating on an SD-Trp screening plate;

12 Culturing at 30℃for 4d.

3. The method for constructing a bifunctional yeast display and secretion system using a 2A peptide strategy as claimed in claim 1, wherein the positive clone selection and extraction are performed by:

randomly selecting 8 clones from the plate, and performing colony PCR;

the 50. Mu.l PCR system was as follows: DNA:1 μl; 1 μl of the upstream primer; 1 μl of the downstream primer; 2 XrapidTaqMasterMix 25 μl; sterile water 22 μl;

the reaction procedure was as follows: 3min at 95 ℃;95 ℃ for 15s; 15s at 50 ℃;72 ℃ for 1min; 5min at 72 ℃ for 35 cycles;

after completion of PCR, 2. Mu.l of each of the samples was aspirated therefrom and subjected to agarose electrophoresis.

4. The method for constructing a bifunctional yeast display and secretion system using a 2A peptide strategy as claimed in claim 1, wherein the specific way of the target protein induced expression is:

1) Collecting all positive clones without mutation, diluting, inoculating into YNB-CAA liquid culture medium containing 2% glucose, shaking culturing at 30deg.C and 225rpm until OD600 = 2-5;

2) Centrifuging at 4000rpm for 5min, and discarding supernatant;

3) Adding YNB-CAA culture medium containing 2% galactose, blowing and mixing, adjusting the culture broth to OD600 = 0.5-1, 30 ℃, culturing at 225rpm, and shaking to induce the expression of target protein;

4) The supernatant and surface displayed proteins were collected.

5. The method for constructing a bifunctional yeast display and secretion system using a 2A peptide strategy as claimed in claim 1, wherein the target protein WesternBlot is identified:

1) Adding appropriate amount of protein into 20 μl protein solution, and boiling at 100deg.C for 5-10min;

2) Centrifuging at 12000rpm for 2min, sucking 20 μl, and performing protein electrophoresis;

3) Cutting PVDF film with proper size, and transferring protein onto the film via wet film transferring process;

4) Washing the membrane for 10min by1 XTBST;

5) Taking a proper amount of sealing liquid to seal the membrane for 10min at room temperature;

6) Washing the membrane for 5min by1 XTBST;

7) The murine anti-GFP is selected as a primary antibody, and incubated for 2 hours at room temperature after dilution;

8) Washing the membrane for 10min by1 XTBST for 3 times;

9) HRP-labeled goat anti-mouse IgG is selected as a secondary antibody, and the secondary antibody is incubated for 45min at room temperature after dilution;

10 1 XTBE for 10min, total 3 times;

11 Taking ECL luminous liquid A, B, uniformly mixing the ECL luminous liquid A, B in equal quantity, dripping the ECL luminous liquid A, B on the front surface of the membrane, and exposing the ECL luminous liquid after incubation;

12 Protein size of interest in the detection of an exoprotein; the size of the protein of interest in display protein assays.