CN117467695B - Method for improving secretion of reporter protein by over-expressing pichia pastoris molecular chaperones - Google Patents
Method for improving secretion of reporter protein by over-expressing pichia pastoris molecular chaperones Download PDFInfo
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Abstract
The invention provides a method for improving secretion of reporter protein by over-expressing pichia pastoris molecular chaperones, which relates to the technical field of biotechnology and comprises the following steps: mCherry-XL was used as a reporter protein; screening the strain with the highest copy and fluorescent protein in the cell aggregation as a starting strain SK01; the MPDI1, PDI1, HAC1, YAP1, kar2 and Ero1 secretion auxiliary factor genes are respectively and over-expressed and integrated into the SK01 strain by CRISPR-Cas9 gene editing technology; the invention screens fluorescent protein as an original strain, genetic modification of a secretion pathway is carried out from the aspect of genetic engineering, and secretion auxiliary factor genes are respectively and over-expressed and integrated into the screened strain through a gene editing technology, so that the secretion capacity of exogenous fluorescent protein is improved; and the transformation system is operated in a PCR eight-connecting tube, and the incubation and heat shock processes are completed in a PCR amplification instrument, so that the experimental operation efficiency is greatly improved.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for improving secretion of reporter protein by over-expressing pichia pastoris chaperones.
Background
Yeast is a strain widely applied to the production of protein medicines, compared with bacteria, yeast cells have certain safety, do not produce endotoxin, have the advantages of post-translational modification, secretion expression, easy genetic operation and the like, greatly reduce the purification cost, currently have more than 5 000 exogenous proteins which can be successfully expressed by pichia pastoris, and along with the secretion expression of more exogenous proteins in the pichia pastoris, many researches find that the intracellular aggregation exists in the secretion expression process of certain exogenous proteins to cause the problems of lower secretion expression quantity, different degrees of degradation of host protease on the exogenous proteins and the like;
The prior art scheme is as follows: the high-efficiency expression of the exogenous protein is realized by optimizing the fermentation process, including the optimization of the components of the culture medium and the induction conditions (parameters such as pH, temperature, time and the like), and the technology has the main defects that: the method for improving the secretion of the reporter protein by over-expressing pichia pastoris chaperones is provided to solve the problems in the prior art.
Disclosure of Invention
Aiming at the problems, the invention provides a method for improving the secretion of reporter protein by over-expressing pichia pastoris molecular chaperones, which is used for screening fluorescent protein as an original strain, carrying out genetic modification of a secretion pathway in the aspect of genetic engineering, and integrating secretion auxiliary factor genes into the screened strain by over-expression respectively through a gene editing technology, thereby improving the secretion capacity of exogenous fluorescent protein.
In order to achieve the purpose of the invention, the invention is realized by the following technical scheme: a method for enhancing secretion of reporter protein by over-expressing pichia pastoris chaperones, comprising the steps of:
S1: mCherry-XL was used as a reporter protein;
s2: screening the strain with the highest copy and fluorescent protein in the cell aggregation as a starting strain SK01;
S3: MPDI1, PDI1, HAC1, YAP1, kar2, ero1 secretion cofactor genes were individually overexpressed and integrated into SK01 strains by CRISPR-Cas9 gene editing techniques.
The further improvement is that: the step S1 comprises the following steps:
s11: optimizing mCherry-XL according to codon preference of Pichia pastoris, wherein the optimized sequence gene is obtained through in vitro total gene synthesis, and is shown as SEQ ID NO. 1;
s12: and constructing a PL405 Pichia pastoris recombinant secretion expression vector.
The further improvement is that: the step S12 includes the steps of:
s121: connecting a Pichia pastoris secretion expression vector pPICZaA with an mCherry-XL gene high-fidelity PCR product obtained in S11 through Gibson assembly;
S122: e.coli TOP10 competent cells are transformed, a mCherry-XL recombinant Pichia pastoris secretion expression vector Gibson assembly is constructed, and a strain for preserving the vector is named PL405;
s123: and selecting a plurality of transformants for sequencing, and preserving the bacterial colony with correct sequencing result.
The further improvement is that: the step S2 comprises the following steps:
S21: linearizing the PL405 vector plasmid through a PCR primer 5'-AOXI(TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTT)、3'-AOXI(CTTAGTTCATCTTGGATGAGATCACGCTTTTGTCATATTAGGTT), recovering the linearization product by using a PCR product purification recovery kit, wherein the PCR primer 5'-AOXI (TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTT) is shown as SEQ ID NO.2, and the PCR primer 3' -AOXI (CTTAGTTCATCTTGGATGAGATCACGCTTTTGTCATATTAGGTT) is shown as SEQ ID NO. 3;
S22: streaking and activating Pichia pastoris wild strain X33 preserved at-80 ℃, culturing for 3 days in a constant temperature incubator at 30 ℃, picking single colony to YPD liquid culture for 10 hours, transferring to 50mLYPD for overnight culture, centrifugally collecting cells when OD600 reaches 0.8-1.0, resuspending the cells with 25mL of sterile water, centrifugally discarding the supernatant, repeating for 1 time, and centrifugally discarding the supernatant; adding 1mL of sterile water to resuspend cells, transferring to a 1.5mL EP tube, centrifuging, and discarding the supernatant; 1mL of 1M LiCl was added to resuspend the cells, and the mixture was left to stand at 30℃for incubation for 1h; centrifuging, removing supernatant, adding 400uL1MLiCl to resuspend cells, and sub-packaging 25uL to a PCR eight-connecting tube to obtain competent cells A;
S23: adding 120uL of 50% PEG4000, 18uL of 1MLiCl, 5uL of 10mg/mL salmon sperm DNA and 2.5ug of DNA to be converted into the competent cells A, mixing the mixture uniformly by vortex, incubating the mixture at the temperature of a PCR amplification instrument for 30min, and then carrying out heat shock at the temperature of 42 ℃ for 20min; removing the supernatant by a centrifugal gun head, adding 0.2mL of YPD resuspended cells, transferring to a 96 shallow well plate, incubating for 2 hours at a constant temperature oscillator of 30 ℃ and 900rpm, coating all the cells on a 0.8mg/mL Zeocin-resistant YPD plate, and culturing for 48 hours in a constant temperature incubator at 30 ℃ to obtain a yeast expression strain of 0.8mg/mL Zeocin;
S24: selecting a plurality of transformants of the obtained yeast expression strain of 0.8mg/mL Zeocin, inoculating the transformants to BMGY liquid medium, carrying out 24-deep-hole plate fermentation, supplementing 3% methanol every 24 hours for induction, fermenting for 96 hours, collecting thalli after fermentation, measuring extracellular and intracellular fluorescence values under the conditions of excitation wavelength 558nm, emission wavelength 590nm and gain 60, and taking PL405-A2 as a starting strain, and naming the starting strain as SK01.
The further improvement is that: the step S3 comprises the following steps:
s31: constructing a gRNA plasmid and a donor plasmid;
s32: preparing competent cells B;
S33: constructing a pichia pastoris secretion auxiliary factor overexpression strain;
S34: and (5) verifying secretion optimization of pichia pastoris.
The further improvement is that: the step S31 includes the steps of:
S311: the sgRNA is designed at benchling on-line websites, and donor left and right homologous arm fragments are obtained through PCR amplification by taking the X33 genome DNA as a template;
S312: regulating each gene by a constitutive promoter PGAP and an inducible promoter PAOX1 respectively, wherein the expression frame is PGAP-gene-TAOX1 or PAOX 1-gene-TAOX;
S313: connecting the left and right homologous arm fragments and the expression frame and the KanaR resistance gene through a Gibson assembly method to construct a donor plasmid;
S314: transforming the gRNA plasmid and the donor plasmid into escherichia coli TOP10 competent cells, picking a plurality of transformants for sequencing, extracting and treating a colony with a correct sequencing result, linearizing the correct donor plasmid, and recovering a linearization product by using a PCR product purification recovery kit.
The further improvement is that: the step S32 includes the steps of:
s321: streaking and activating recombinant bacteria SK01 preserved at-80 ℃, culturing for 3d in a constant temperature incubator at 30 ℃, picking single colony to YPD liquid culture for 10h, and transferring to 50mLYPD for overnight culture;
s322: when the OD600 reaches 0.8-1.0, cells are collected by centrifugation, 25mL of sterile water is used for resuspension, supernatant is discarded by centrifugation, and the supernatant is discarded by centrifugation for 1 time;
S323: adding 1mL of sterile water to resuspend cells, transferring to a 1.5mL EP tube, centrifuging, and discarding the supernatant;
s324: 1mL of 1MLiCl was added to resuspend the cells, and the mixture was left to stand at 30℃for incubation for 1h;
S325: the supernatant was centrifuged off, 400uL of 1MLiCl was added to resuspend the cells, and 25uL was dispensed into PCR octants to obtain competent cell B.
The further improvement is that: the step S33 includes the steps of:
S331: adding competent cell B into 120 mu L of 50% PEG4000, 18 mu L of 1MLiCl and 5 mu L of 10mg/mL salmon sperm DNA, respectively taking 1ug of gRNA plasmid of each gene and donor plasmid linearization fragment, adding into a transformation system, and mixing uniformly by vortex;
S332: incubating for 30min at 30 ℃ in a PCR amplification instrument, and then carrying out heat shock for 20min at 42 ℃;
S333: the supernatant was removed by a centrifuge gun head, 0.2mL of YPD resuspended cells were added, transferred to a 96 shallow well plate, incubated at a constant temperature shaker of 30℃and 900rpm for 2 hours, and all coated on a 0.1mg/mL Zeocin-resistant YPD plate, and cultured in a constant temperature incubator of 30℃for 96 hours to obtain a 0.1mg/mL Zeocin recombinant strain.
The further improvement is that: the step S34 includes the steps of:
s341: in the obtained yeast recombinant strain with the concentration of 0.1mg/mL Zeocin, selecting a plurality of transformants for colony PCR verification;
S342: inoculating a plurality of transformants of each colony which is verified successfully to a BMGY liquid culture medium, fermenting by a 24 deep-hole plate for 96 hours, and collecting thalli after fermentation;
S343: under the conditions of excitation wavelength 558nm, emission wavelength 590nm and gain 60, the SK01 is used as a control to measure the extracellular and intracellular fluorescence values.
The beneficial effects of the invention are as follows:
1. The invention screens fluorescent protein as an initial strain, genetic modification of a secretion pathway is carried out in terms of genetic engineering, and secretion auxiliary factor genes are respectively and over-expressed and integrated into the screened strain through a gene editing technology, so that the secretion capacity of the exogenous fluorescent protein is improved.
2. The transformation system is completed in the PCR eight-connecting-tube operation and incubation and heat shock process in the PCR amplification instrument, so that the experimental operation efficiency is greatly improved, the experimental method is simplified, and the reagent cost is saved.
Drawings
FIG. 1 is a diagram showing the result of PL405 sequencing and comparison in the present invention;
FIG. 2 is a diagram showing the results of screening of the starting strain according to the present invention;
FIG. 3 is a diagram showing the PCR results of the over-expressed secreted cofactor gene of the present invention integrated into the SK01 genomic colony, wherein lane M is the DL 5000 standard molecular weight Marker; all CK negative controls have a size of 2.5k, and positive transformants have a stripe size of 5k, wherein 1 and 2 represent that two transformants are selected when the constructed strain is fermented, namely two biological repeats, and a gel diagram is obtained by carrying out PCR identification on the two biological repeats of the constructed strain;
FIG. 4 is a graph showing the results of optimization of the secretory pathway of Pichia pastoris according to the present invention.
Detailed Description
The present invention will be further described in detail with reference to the following examples, which are only for the purpose of illustrating the invention and are not to be construed as limiting the scope of the invention.
Example 1
According to fig. 1,2, 3 and 4, the present embodiment provides a method for improving secretion of reporter protein by over-expressing pichia pastoris chaperones, comprising the following steps:
S1: mCherry-XL was used as a reporter protein;
s2: screening the strain with the highest copy and fluorescent protein in the cell aggregation as a starting strain SK01;
S3: MPDI1, PDI1, HAC1, YAP1, kar2, ero1 secretion cofactor genes were individually overexpressed and integrated into SK01 strains by CRISPR-Cas9 gene editing techniques.
The method specifically comprises the following steps:
A starting strain is constructed by the following steps: optimizing a fluorescent protein gene mCherry-XL codon; constructing a pichia pastoris secretion expression vector pPICZaA-mCherry-XL; single exchange integration into pichia wild type X33; screening 0.8mg/mL Zeocin high copy strain; fermenting with 24-hole plates; measuring fluorescence value by excitation wavelength 558nm, emission wavelength 590nm and gain 60; screening target strain as original strain.
An optimization method suitable for secretory expression of exogenous proteins. The method comprises the following steps: on the basis of original strains, secretion auxiliary factors such as folding enzyme genes MPDI1, PDI1, HAC1, transcription factor gene YAP1, molecular chaperone genes Kar2, ero1 and the like are selected, and the genes represent the secretion expression effect of foreign proteins under the regulation and control of a strong constitutive promoter (PGAP) and a strong inducible promoter (PAOX 1) which are commonly used in pichia pastoris.
The secretory pathway optimization of pichia pastoris is carried out from the over-expressed secretory auxiliary factors based on the genetic engineering technology, the modulation and control intensities are regulated by different promoter signs based on the over-expressed secretory auxiliary factors, and the transformation method is optimized, so that the capacity of carrying out pichia pastoris transformation in a large scale is improved.
Example two
According to fig. 1,2, 3 and 4, the present embodiment provides a method for improving secretion of reporter protein by over-expressing pichia pastoris chaperones, comprising the following steps:
The secretory pathway optimizing effect is explored, and a convenient and quick reporting system is established. Fluorescent proteins have been widely used in the research of mammals, bacteria and fungi because of their small molecular weight, strong and stable fluorescent expression, and thus mCherry-XL is used as a reporter protein.
The mCherry-XL is optimized according to the codon preference of Pichia pastoris, and the optimized sequence gene is obtained through in vitro total gene synthesis. The optimized sequence is shown in a sequence table.
Constructing a PL405 Pichia pastoris recombinant secretion expression vector: and (3) connecting the Pichia pastoris secretion expression vector pPICZaA with the mCherry-XL gene high-fidelity PCR product obtained in the step, transforming the escherichia coli TOP10 competent cells, and constructing the mCherry-XL recombinant Pichia pastoris secretion expression vector Gibson assembly, wherein the strain for preserving the vector is named PL405. Three transformants were picked for sequencing, the results of the sequencing comparison are shown in FIG. 1, and the correct colonies of the sequencing results were kept.
To better characterize the effect of over-expressed secretion cofactors on the secretory expression of pichia pastoris fluorescent protein, a strain with high copy number and intracellular aggregation of fluorescent protein is selected as an initial strain.
Linearizing the PL405 vector plasmid through a PCR primer 5'-AOXI(TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTT) 、3'-AOXI(CTTAGTTCATCTTGGATGAGATCACGCTTTTGTCATATTAGGTT), recovering the linearization product by using a PCR product purification recovery kit, wherein the PCR primer 5'-AOXI (TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTT) is shown as SEQ ID NO.2, and the PCR primer 3' -AOXI (CTTAGTTCATCTTGGATGAGATCACGCTTTTGTCATATTAGGTT) is shown as SEQ ID NO. 3;
preparation of competent cells: streaking and activating Pichia pastoris wild strain X33 preserved at-80 ℃, culturing for 3 days in a constant temperature incubator at 30 ℃, picking single colony to YPD liquid culture for 10 hours, transferring to 50mLYPD for overnight culture, centrifugally collecting cells when OD600 reaches 0.8-1.0, resuspending the cells with 25mL of sterile water, centrifugally discarding the supernatant, repeating for 1 time, and centrifugally discarding the supernatant; adding 1mL of sterile water to resuspend cells, transferring to a 1.5mL EP tube, centrifuging, and discarding the supernatant; 1mL of 1M LiCl is added to resuspend the cells, and the mixture is kept stand and incubated for 1h at 30 ℃; the supernatant was centrifuged off, 400uL of 1M LiCl was added to resuspend the cells, and 25uL was dispensed into PCR octants.
Construction of PL405 pichia pastoris secretory expression strain and screening of high copy strains: adding 120uL 50% PEG4000, 18uL 1M LiCl, 5uL 10mg/mL salmon sperm DNA and 2.5ug DNA to be converted into competent cells in the steps, mixing uniformly by vortex, incubating for 30min at 30 ℃ in a PCR amplification instrument, and then carrying out heat shock for 20min at 42 ℃; the supernatant was removed by centrifugation at the tip, 0.2mL of YPD resuspended cells were added, transferred to a 96 shallow well plate, incubated at 30℃with a constant temperature shaker at 900rpm for 2h, plated on 0.8mg/mL Zeocin-resistant YPD plates, and incubated in a constant temperature incubator at 30℃for 48h.
Screening of the starting strain: 8 transformants of the 0.8mg/mL Zeocin yeast expression strain obtained in the above steps are selected and inoculated in BMGY liquid culture medium, 24 deep-hole plate fermentation is carried out, 3% methanol is fed every 24 hours for induction, fermentation is carried out for 96 hours, after fermentation, bacterial cells are collected, extracellular and intracellular fluorescence values are measured under the conditions of excitation wavelength 558nm, emission wavelength 590nm and gain 60, the analysis result of the fluorescence values is shown in figure 2, PL405-A2 is taken as an initial strain, and the initial strain is named SK01.
MPDI1, PDI1, HAC1, YAP1, kar2, ero1 secretion cofactor genes were individually overexpressed and integrated into SK01 strains by CRISPR-Cas9 gene editing techniques.
Chaperones Kar2 residing in the endoplasmic reticulum bind to nascent polypeptide chains and assist protein folding by shielding the unfolded region from interaction with adjacent proteins. Kar2 belonging to the family of HSP70 chaperones acts mainly on nascent polypeptide chains and promotes their transport to the endoplasmic reticulum.
Construction of the gRNA plasmid and the donor plasmid: the sgRNA is designed on benchling online websites, about 1kb homologous arm fragments of a donor are obtained by taking X33 genome DNA as a template and amplifying by PCR, each gene is regulated and controlled by a constitutive promoter PGAP and an inducible promoter PAOX1, an expression frame is PGAP-gene-TAOX1 or PAOX1-gene-TAOX1, the about homologous arm fragments, the expression frame and a Kanar resistance gene are connected by a Gibson assembly method to construct a donor plasmid, the gRNA plasmid and the donor plasmid transform competent cells of the escherichia coli TOP10, three transformants are picked and sequenced, a colony with a correct sequencing result is extracted and treated, the correct donor plasmid is linearized, and a linearized product is recovered by using a PCR product purification recovery kit.
Preparation of competent cells: streaking and activating recombinant bacteria SK01 preserved at-80 ℃, culturing for 3 days in a constant temperature incubator at 30 ℃, picking single colony to YPD liquid for culturing for 10 hours, transferring to 50mLYPD for overnight culture, centrifugally collecting cells when OD600 reaches 0.8-1.0, centrifugally suspending the cells with 25mL of sterile water, centrifugally discarding the supernatant, repeating for 1 time, centrifugally discarding the supernatant; adding 1mL of sterile water to resuspend cells, transferring to a 1.5mL EP tube, centrifuging, and discarding the supernatant; 1mL of 1M LiCl is added to resuspend the cells, and the mixture is kept stand and incubated for 1h at 30 ℃; the supernatant was centrifuged off, 400uL1M LiCl was added to resuspend the cells, and 25uL was dispensed into PCR octants.
Construction of Pichia pastoris secretion cofactor overexpression strain: adding 120 mu L of 50% PEG4000, 18 mu L of 1M LiCl and 5 mu L of 10mg/mL salmon sperm DNA into competent cells in the steps, respectively taking 1ug of gRNA plasmid of each gene and donor plasmid linearization fragment, adding into a transformation system, vortex mixing uniformly, incubating for 30min at the temperature of a PCR amplification instrument at the temperature of 30 ℃, and then carrying out heat shock for 20min at the temperature of 42 ℃; the supernatant was removed by centrifugation at the tip, 0.2mL of YPD resuspended cells were added, transferred to a 96 shallow well plate, incubated at 30℃with a constant temperature shaker at 900rpm for 2h, plated on 0.1mg/mL Zeocin-resistant YPD plates, and incubated in a constant temperature incubator at 30℃for 96h.
The Pichia pastoris secretion optimization method is verified: the 0.1mg/mL Zeocin yeast recombinant strain obtained in the above steps is picked up to obtain transformants, colony PCR verification is carried out, the successfully verified colonies are respectively picked up to obtain two transformants, each transformant is inoculated to BMGY liquid culture medium as shown in figure 3, 24 deep-hole plate fermentation is carried out for 96 hours, after fermentation is finished, bacterial cells are collected, extracellular and intracellular fluorescence values are measured under the conditions of excitation wavelength 558nm, emission wavelength 590nm and gain 60, SK01 is used as a control, strain information is shown in the following table 1, and the analysis results of the fluorescence values are shown in figure 4.
TABLE 1 Pichia secretory pathway optimization recombinant Strain information
Description: examples SK01/fp1+fp7a represent SK02 strain was constructed by linearizing SK01 as starting strain, FP1 as sgRNA plasmid, FP7 as donor plasmid by CRISPR-Cas9 gene editing technique, and a represents donor plasmid.
Wherein, mCherry-XL gene optimized sequence:
ATGGTTTCTAAAGGCGAAGAGGATAATATGGCGATTATTAAAGAATTCATGAGATTTAAGGTTCATATGGAAGGTTCCGTTAACGGGCATGAATTTGAAATTGAAGGGGAAGGGGAAGGACGTCCTTATGAAGGTACCCAAACAGCAAAGCTGAAAGTTACAAAAGGTGGTCCGCTTCCCTTTGCTTGGGATATCCTGAGCCCCCAGTTTATGTACGGCAGCAAAGCCTATGTCAAGCATCCAGCAGACATTCCTGATTATCTAAAACTCAGTTTTCCTGAAGGATTTAAATGGGAACGTGTGATGAATTTTGAGGATGGCGGCGTTGTCACCGTGACCCAGGATTCAAGCCTACAGGACGGTGAGTTTATTTATAAAGTAAAACTGCGGGGTACCAATTTCCCGTCTGATGGCCCGGTCATGCAAAAAAAAACGATGGGTAGCGAAGCCAGCAGTGAAAGGATGTATCCGGAAGATGGCGCGCTGAAAGGAGAAGTGAAATACCGTCTGAAGCTGAAAGATGGAGGACATTATGATGCAGAAGTTAAAACAACATATAAGGCAAAAAAGCCGGTTCAGCTGCCGGGAGCATATAACGTCAACCGTAAACTTGATATCACAAGCCACAATGAAGACTATACGATTGTTGAGCAGTATGAGAGAGCTGAAGGCCGCCATTCAACCGGCGGAATGGATGAATTATATAAA.(SEQ ID NO.1)
The mCherry-XL has the main function of serving as a fluorescent protein molecular beacon to characterize the optimization effect of the secretion pathway of pichia pastoris, and the mCherry-XL fluorescent protein molecular beacon can be intuitively and rapidly subjected to experimental screening.
PCR primer:
5' -AOXI (TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTT) is shown as SEQ ID NO. 2;
3' -AOXI (CTTAGTTCATCTTGGATGAGATCACGCTTTTGTCATATTAGGTT) is shown as SEQ ID NO. 3.
The primer is used for amplifying the successfully constructed PL405 plasmid and the required DNA fragment by PCR, and is prepared for the next Pichia pastoris conversion experiment.
Verification example:
As shown in Table 2, according to the analysis of experimental results, compared with the control SK01, the secretion capacity of the exogenous fluorescent protein is improved by 27%, the intracellular fluorescent expression quantity is reduced by 25%, and the regulatory capacity of the pAOX1 inducible promoter is stronger than that of the constitutive promoter PGAP (SK 06).
TABLE 2 overexpression of chaperone gene Kar2 optimization of secretion levels
| Strain numbering | FL/OD 600 (supernatant) | FL/OD 600 (precipitation) |
| SK01 | 13793±670.34 | 18209±1262.89 |
| SK06 | 15633±521.14 | 17112±2666.50 |
| SK07 | 18946±672.46 | 14532±3721.50 |
Note that: FL/OD 600 is the mean.+ -. Standard deviation of two independent replicates.
The method for improving the secretion of the reporter protein by over-expressing the pichia pastoris molecular chaperones screens fluorescent protein as an initial strain, genetic modification of a secretion pathway is carried out from the aspect of genetic engineering, and secretion auxiliary factor genes are respectively and over-expressed and integrated into the screened strain by a genetic editing technology, so that the secretion capacity of the exogenous fluorescent protein is improved. The transformation system is completed in the PCR eight-connecting-tube operation and incubation and heat shock process in the PCR amplification instrument, so that the experimental operation efficiency is greatly improved, the experimental method is simplified, and the reagent cost is saved.
The foregoing has shown and described the basic principles, principal features and advantages of the invention. It will be understood by those skilled in the art that the present invention is not limited to the embodiments described above, and that the above embodiments and descriptions are merely illustrative of the principles of the present invention, and various changes and modifications may be made without departing from the spirit and scope of the invention, which is defined in the appended claims. The scope of the invention is defined by the appended claims and equivalents thereof.
Claims (6)
1. The method for improving the secretion of the reporter protein by over-expressing the pichia pastoris molecular chaperone is characterized by comprising the following steps:
S1: the mCherry-XL is used as a reporter protein, the mCherry-XL gene sequence is optimized according to the codon preference of pichia pastoris, and the optimized gene sequence is obtained through in vitro total gene synthesis and is shown as SEQ ID NO. 1; constructing a PL405 pichia pastoris recombinant secretion expression vector, connecting a pichia pastoris secretion expression vector pPICZaA with a mCherry-XL gene high-fidelity PCR product obtained in S1, transforming an escherichia coli TOP10 competent cell, constructing a mCherry-XL recombinant pichia pastoris secretion expression vector, naming the vector as PL405, selecting a plurality of transformants for sequencing, and preserving a colony with a correct sequencing result;
S2: linearizing a PL405 vector plasmid through a PCR primer 5'-AOXI(TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTT)、3'-AOXI(CTTAGTTCATCTTGGATGAGATCACGCTTTTGTCATATTAGGTT), recovering a linearization product by using a PCR product purification recovery kit, wherein the PCR primer 5'-AOXI (TTTGGTTCGTTGAAATGCTAACGGCCAGTTGGTCAAAAAGAAACTT) is shown as SEQ ID NO.2, the PCR primer 3' -AOXI (CTTAGTTCATCTTGGATGAGATCACGCTTTTGTCATATTAGGTT) is shown as SEQ ID NO.3, streaking and activating a Pichia pastoris wild strain X33 preserved at-80 ℃, culturing for 3 days in a constant temperature incubator at 30 ℃, picking up a single colony to YPD liquid culture for 10 hours, transferring to 50mLYPD for overnight culture, centrifugally collecting cells when the OD600 reaches 0.8-1.0, centrifugally discarding the supernatant, repeatedly carrying out 1 time, centrifugally discarding the supernatant; adding 1mL of sterile water to resuspend cells, transferring to a 1.5mL EP tube, centrifuging, and discarding the supernatant; 1mL of 1M LiCl was added to resuspend the cells, and the mixture was left to stand at 30℃for incubation for 1h; centrifuging, removing supernatant, adding 400uL1MLiCl to resuspend cells, subpackaging 25uL to a PCR octamer tube to obtain competent cells A, adding 120uL 50% PEG4000, 18uL 1MLiCl, 5uL 10mg/mL salmon sperm DNA and 2.5ug of DNA to be converted, mixing uniformly by vortex, incubating for 30min at 30 ℃ in a PCR amplification instrument, and then carrying out heat shock for 20min at 42 ℃; removing supernatant by a centrifugal gun head, adding 0.2mL YPD heavy suspension cells, transferring to a 96 shallow hole plate, incubating for 2 hours at a constant temperature oscillator of 30 ℃, coating all the cells on a 0.8mg/mL Zeocin-resistant YPD plate, culturing for 48 hours in a constant temperature incubator at 30 ℃, picking a plurality of transformants of the obtained 0.8mg/mL Zeocin yeast expression strain, inoculating the transformants to a BMGY liquid culture medium, fermenting for 24 deep hole plates, supplementing 3% methanol every 24 hours for induction, fermenting for 96 hours, collecting thalli, measuring extracellular and intracellular fluorescence values under conditions of excitation wavelength of 558nm, emission wavelength of 590nm and gain of 60, and screening the strain with the highest copy and fluorescent protein concentrated in the cells as a starting strain SK01;
s3: the Kar2 gene is integrated into the SK01 strain through CRISPR-Cas9 gene editing technology.
2. The method of claim 1, wherein the method of enhancing secretion of a reporter protein comprises over-expressing a pichia pastoris chaperone: the step S3 comprises the following steps:
s31: constructing a gRNA plasmid and a donor plasmid;
s32: preparing competent cells B;
s33: constructing a pichia pastoris Kar2 overexpression strain;
S34: and (5) verifying secretion optimization of pichia pastoris.
3. The method of enhancing secretion of reporter protein by over-expressing pichia pastoris chaperones of claim 2, wherein: the step S31 includes the steps of:
S311: the sgRNA is designed at benchling on-line websites, and donor left and right homologous arm fragments are obtained through PCR amplification by taking the X33 genome DNA as a template;
S312: regulating and controlling Kar2 genes by a constitutive promoter PGAP and an inducible promoter PAOX1 respectively, wherein an expression frame is PGAP-Kar2-TAOX1 or PAOX1-Kar2-TAOX1;
S313: connecting the left and right homologous arm fragments and the expression frame and the KanaR resistance gene through a Gibson assembly method to construct a donor plasmid;
s314: transforming the gRNA plasmid and the donor plasmid into escherichia coli TOP10 competent cells, picking a plurality of transformants for sequencing, extracting plasmids from colonies with correct sequencing results, linearizing the correct donor plasmid, and recovering linearization products by using a PCR product purification recovery kit.
4. The method of claim 3, wherein the method of enhancing secretion of a reporter protein comprises over-expressing a pichia pastoris chaperone: the step S32 includes the steps of:
s321: streaking and activating recombinant bacteria SK01 preserved at-80 ℃, culturing for 3d in a constant temperature incubator at 30 ℃, picking single colony to YPD liquid culture for 10h, and transferring to 50mLYPD for overnight culture;
s322: when the OD600 reaches 0.8-1.0, cells are collected by centrifugation, 25mL of sterile water is used for resuspension, supernatant is discarded by centrifugation, and the supernatant is discarded by centrifugation for 1 time;
S323: adding 1mL of sterile water to resuspend cells, transferring to a 1.5mL EP tube, centrifuging, and discarding the supernatant;
s324: 1mL of 1MLiCl was added to resuspend the cells, and the mixture was left to stand at 30℃for incubation for 1h;
S325: the supernatant was centrifuged off, 400uL of 1MLiCl was added to resuspend the cells, and 25uL was dispensed into PCR octants to obtain competent cell B.
5. The method for improving secretion of reporter protein by over-expressing pichia pastoris chaperones according to claim 4, wherein: the step S33 includes the steps of:
S331: adding competent cell B into 120 mu L of 50% PEG4000, 18 mu L of 1MLiCl and 5 mu L of 10mg/mL salmon sperm DNA, respectively taking 1ug of gRNA plasmid and donor plasmid linearization fragment, adding into a transformation system, and mixing by vortex;
S332: incubating for 30min at 30 ℃ in a PCR amplification instrument, and then carrying out heat shock for 20min at 42 ℃;
S333: the supernatant was removed by a centrifuge gun head, 0.2mL of YPD resuspended cells were added, transferred to a 96 shallow well plate, incubated at a constant temperature shaker of 30℃and 900rpm for 2 hours, and all coated on a 0.1mg/mL Zeocin-resistant YPD plate, and cultured in a constant temperature incubator of 30℃for 96 hours to obtain a 0.1mg/mL Zeocin recombinant strain.
6. The method for improving secretion of reporter protein by over-expressing pichia pastoris chaperones according to claim 5, wherein: the step S34 includes the steps of:
s341: in the obtained yeast recombinant strain with the concentration of 0.1mg/mL Zeocin, selecting a plurality of transformants for colony PCR verification;
S342: inoculating a plurality of transformants of each colony which is verified successfully to a BMGY liquid culture medium, fermenting by a 24 deep-hole plate for 96 hours, and collecting thalli after fermentation;
S343: under the conditions of excitation wavelength 558nm, emission wavelength 590nm and gain 60, the SK01 is used as a control to measure the extracellular and intracellular fluorescence values.
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| 改良毕赤酵母分泌表达外源蛋白能力的研究进展;关波;金坚;李华钟;;微生物学报;20110704(07);851-857 * |
| 毕赤酵母Kar2p过表达对假黑盘菌素表达量的影响;王楠;李刚强;郭文芳;刘德虎;;生物技术通报;20160126(01);180-186 * |
| 毕赤酵母高效表达外源蛋白 的分子水平策略;张欣然等;食品与发酵工业;20160126;第48卷(第17期);321-328 * |
| 过表达KAR2基因对毕赤酵母产米黑根毛霉脂肪酶的影响;黄金金;孙梦雪;王一洲;赵庆伊;张馨;张恺倪;;安徽农业科学;20200718(14);103-107 * |
| 酵母分泌系统工程化改造研究进展;常亮;刘晓志;孟雅娟;马志;高健;;生物技术进展;20130525(03);185-189 * |
| 雅致放射毛霉羧肽酶Y在毕赤酵母中的高效分泌表达;张欢欢;陈昶旭;刘中美;周哲敏;;食品与发酵工业;20180420(11);1-6 * |
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Denomination of invention: Method for Overexpressing Pichia pastoris Molecular Companion to Enhance Reporter Protein Secretion Granted publication date: 20240503 Pledgee: China Construction Bank Co.,Ltd. Nanjing Gaochun sub branch Pledgor: Nanjing hongruijie Biomedical Technology Co.,Ltd. Registration number: Y2024980040076 |