CN115141840A - Yeast optimized technological preparation method - Google Patents
Yeast optimized technological preparation method Download PDFInfo
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- CN115141840A CN115141840A CN202210793497.1A CN202210793497A CN115141840A CN 115141840 A CN115141840 A CN 115141840A CN 202210793497 A CN202210793497 A CN 202210793497A CN 115141840 A CN115141840 A CN 115141840A
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/80—Vectors or expression systems specially adapted for eukaryotic hosts for fungi
- C12N15/81—Vectors or expression systems specially adapted for eukaryotic hosts for fungi for yeasts
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- C12N2800/00—Nucleic acids vectors
- C12N2800/22—Vectors comprising a coding region that has been codon optimised for expression in a respective host
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- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/645—Fungi ; Processes using fungi
- C12R2001/84—Pichia
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Abstract
The invention discloses a yeast optimized process preparation method, which comprises the following steps: predicting a target gene signal peptide, removing a signal peptide sequence, carrying out codon optimization according to a pichia pastoris expression system, and carrying out gene synthesis to pPICZ alpha A; predicting target gene signal peptide by using SignalP 4.0 and SignalP5.1; when codon optimization is carried out on a pichia pastoris expression system, the SacI enzyme cutting site is avoided; when the gene is synthesized to pPICZ alpha A, the target gene is close to the carrier alpha-factor, and 6 × His is arranged on the C-terminal band; the invention overcomes the defects of an escherichia coli expression system through yeast expression, can be quickly propagated and fermented at high density, and can carry out post-translational modification and processing on protein. Secondly, the target protein is secreted into the culture medium, and the subsequent purification process is greatly simple and convenient. Finally, the protein expression of eukaryote is suitable, the human epidermal growth factor is eukaryotic protein, and the yeast expression can improve the yield and reduce the cost.
Description
Technical Field
The invention belongs to the technical field of optimized yeast, and particularly relates to a yeast optimized process preparation method.
Background
The protein expressed by escherichia coli is not modified and does not necessarily have natural activity, an expression system cannot regulate and control expression time and expression level, non-physiological reaction can be caused by over-expression, the protein is often expressed in an inclusion body form at present, so that the purification is difficult, the operation flow of the whole preparation method is complex, the yield of an expression product is low, and therefore the yeast optimization process preparation method is provided.
Disclosure of Invention
The invention aims to provide a yeast optimized process preparation method to solve the problems in the background technology.
In order to achieve the purpose, the invention provides the following technical scheme: a process for preparing yeast includes such steps as optimizing the yeast,
constructing a vector: predicting a target gene signal peptide, removing a signal peptide sequence, carrying out codon optimization according to a pichia pastoris expression system, and carrying out gene synthesis to pPICZ alpha A; predicting target gene signal peptide by using SignalP 4.0 and SignalP5.1;
when codon optimization is carried out on a pichia pastoris expression system, the SacI enzyme cutting site is avoided;
when the gene was synthesized to pPICZ. Alpha.A, the gene of interest was placed next to the vector alpha-factor, with 6 × His in the C-terminal band.
Preferably, pichia pastoris is transformed into plasmid shape by electricity;
preferably, the target vector is linearized;
the linearization of the target vector comprises the following steps:
(1) The vector restriction enzyme system was formulated as follows:
| components | Volume (ul) |
| Plasmid (5-10 ug) | |
| 10× |
5 |
| SacI | 1ul |
| Alkaline phosphatase | 1ul |
| ddH 2 O | Supplement to 50 |
(2) Enzyme digestion is carried out at 37 ℃ overnight;
(3) Detecting by agarose gel electrophoresis, and taking plasmid which is not cut by enzyme as a reference;
(4) After the enzyme digestion is detected successfully, the enzyme is inactivated at 65 ℃ for 20 min.
Preferably, the linearized vector is purified and recovered;
wherein, the linearized vector is purified and recovered; the method comprises the following steps:
(1) The carrier purification system was configured as follows:
| components | Volume (ul) |
| |
50 |
| Nucleic |
10 |
| 3M NaAc，pH＝5.2 | 6 |
| |
165 |
(2) Standing at-20 deg.C for more than 35 min;
(3) Centrifuging at 12000rpm at 4 deg.C for 15min, and discarding the supernatant, wherein white precipitate on the wall can be observed;
(4) Adding 400ul of precooled 80% ethanol for resuspension and precipitation;
(5) Centrifuging at 12000rpm at 4 deg.C for 10min, removing supernatant, and drying;
(6) The precipitate was dissolved by adding 10ul ddH 2O.
Preferably, yeast electroporation competes are prepared;
wherein, the preparation of the yeast electrotransformation competence comprises the following steps:
the first day:
(1) Adding 5ml YPD into a 50ml centrifuge tube, inoculating X-33 strain, and culturing at 30 ℃ overnight;
the following day:
(2) Transferring 50ul of the bacterial liquid to a 250ml conical flask of 50ml YPD after 10-12h, and culturing overnight until OD600=1.3-1.5;
and (3) on the third day:
(3) Centrifuging at 4000rpm at 4 deg.C for 5min, resuspending with 10ml buffera, water bathing at 30 deg.C for 15min, and adding pre-cooled sterilized water to 50ml;
buffer A:20mlYPD +2ml2MHEPES (pH =8.0, filter sterilized) +0.5ml1MDTT (filter sterilized);
(4) Centrifuging at 4000rpm at 4 deg.C for 5min, and resuspending with 50ml of pre-cooled sterile water (containing 0.3ml of 2MHEPES, pH = 8.0);
(5) Centrifuging at 4000rpm at 4 ℃ for 5min and resuspending with 4ml of precooled 1 Msorbitol;
(6) Centrifugation is carried out at 4000rpm for 5min at 4 ℃, then 100ul of precooled 1Msorbitol is used for resuspension, the bacterial liquid is viscous at this time, 80 ul/tube split charging is carried out, and the mixture is placed on ice.
Preferably, yeast is electrotransferred via a linearized vector;
wherein, the yeast electrotransformation by a linearized vector comprises the following steps:
(1) Taking 80ul of competent cells, adding 6ug of linearized pPICZ alpha A-gene, uniformly mixing, and transferring to a precooled 0.2cm electric shock cup;
(2) Standing on ice for 5min;
(3) According to the yeast electric shock parameter setting (1.5kV, 25uF,200 omega), electric shock is carried out;
(4) 2ml of pre-cooled 1M sorbent + HEPES (10ml 1M sorbent +100ul 2M HEPES, pH = 8.0) was immediately added and transferred to a 2ml sterile centrifuge tube;
(5) Standing and incubating for 1-2h at 30 ℃;
(6) After dilution 5-fold, 10-fold, and 100-fold, the cells were plated at 300 ul/plate on 15cm YPD plates containing 100mg/l Zeocin, and cultured at 30 ℃ until colonies grew.
Preferably, transformants are screened;
wherein, the screening of the transformant comprises the following steps:
(1) Randomly selecting 24 clones from the plate, and carrying out colony PCR; (colony PCR uses a rapid amplification yeast cloning kit independently developed by the company, the product number is RY 8001);
(2) 2 of them were selected for sequencing, and 7 clones were retained for use after correct alignment.
Preferably, the transformant expresses the pilot;
wherein, the transformant expression small test comprises the following steps:
(1) Preparing 7 50ml conical flasks, respectively adding 5ml YPG medium, respectively inoculating the above clones verified to be correct, and culturing at 30 deg.C and 220rpm for 1-2 days until the bacterial liquid is saturated;
YPG medium: 1% yeast extract, 2% peptone, 1% glycerol;
(2) Transferring the mixture into a 50ml centrifuge tube, centrifuging the mixture for 5min at 4000rpm, and removing supernatant;
(3) Resuspending the cells in 5ml BMMY medium, transferring to a new sterile 50ml conical flask, adding methanol to a final concentration of 0.5%, and culturing at 28 ℃ and 220rpm for 6 days;
BMMY medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source, 100mMPBS buffer (pH = 5.7);
(4) Adding methanol (the final concentration is 0.5%) every 24 h;
(5) And collecting bacteria liquid in the morning of 6d, centrifuging at 4000rpm for 5min, and collecting supernatant for expression identification.
Preferably, the transformant amplifies expression.
Wherein the method for expanding the expression of the transformant comprises the following steps:
(1) Adding 50ml of YPG culture medium into a 250ml conical flask, inoculating an optimal expression transformant, and culturing at 30 ℃ and 220rpm for 1-2 days until the strain liquid is saturated;
(2) Transferring the mixture into a 50ml centrifuge tube, centrifuging the mixture for 5min at 4000rpm, and removing supernatant;
(3) Resuspending the cells in 50ml BMMY medium, transferring to a new sterile 250ml conical flask, adding methanol to a final concentration of 0.5%, and culturing at 28 ℃ and 220rpm for 6d;
(4) Adding methanol (the final concentration is 0.5%) every 24 h;
(6) And collecting bacterial liquid in the morning at 6d, centrifuging at 4000rpm for 5min, and collecting supernatant for expression identification.
Compared with the prior art, the invention has the beneficial effects that:
the invention overcomes the defects of an escherichia coli expression system through yeast expression, can be quickly propagated and fermented at high density, and can carry out post-translational modification and processing on protein. Secondly, the target protein is secreted into the culture medium, and the subsequent purification process is greatly simple and convenient. Finally, the protein expression of eukaryote is suitable, the human epidermal growth factor is eukaryotic protein, and the yeast expression can improve the yield and reduce the cost.
Drawings
FIG. 1 is a schematic diagram of ingredients of a vector enzyme digestion system of the invention;
FIG. 2 is a schematic representation of the formulation of the carrier purification system of the present invention;
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
Referring to fig. 1 and fig. 2, the present invention provides a technical solution:
a process for preparing yeast includes such steps as optimizing the yeast,
constructing a vector: predicting a target gene signal peptide, removing a signal peptide sequence, carrying out codon optimization according to a pichia pastoris expression system, and carrying out gene synthesis to pPICZ alpha A; predicting target gene signal peptide by using SignalP 4.0 and SignalP5.1;
when codon optimization is carried out on a pichia pastoris expression system, the SacI enzyme cutting site is avoided;
when the gene was synthesized to pPICZ. Alpha.A, the gene of interest was located next to the vector alpha-factor, with 6 XHis at the C-terminal band.
In this embodiment, preferably, pichia pastoris is transformed into plasmid form by electroporation;
in this embodiment, it is preferable that the objective vector is linearized;
the linearization of the target vector comprises the following steps:
(1) The vector restriction enzyme system was formulated as follows:
| components | Volume (ul) |
| Plasmid (5-10 ug) | 6ug |
| 10× |
5 |
| SacI | 1ul |
| Alkaline phosphatase | 1ul |
| ddH 2 O | Supplement to 50 |
(2) Enzyme digestion is carried out at 37 ℃ overnight;
(3) Detecting by agarose gel electrophoresis, and taking plasmid which is not cut by enzyme as a reference;
(4) After enzyme digestion is detected successfully, inactivation is carried out for 20min at 65 ℃.
In this embodiment, preferably, the linearized vector is purified and recovered;
wherein, the linearized vector is purified and recovered; the method comprises the following steps:
(1) The carrier purification system was configured as follows:
| components | Volume (ul) |
| |
50 |
| Nucleic |
10 |
| 3M NaAc，pH＝5.2 | 6 |
| |
165 |
(2) Standing at-20 deg.C for over 35 min;
(3) Centrifuging at 12000rpm at 4 deg.C for 15min, and discarding the supernatant, wherein white precipitate on the wall can be observed;
(4) Adding 400ul of precooled 80% ethanol for resuspension and precipitation;
(5) Centrifuging at 12000rpm at 4 deg.C for 10min, removing supernatant, and drying;
(6) The precipitate was dissolved by adding 10ul ddH 2O.
In this embodiment, it is preferable to prepare yeast for electroporation competence;
wherein, the preparation of the yeast electrotransformation competence comprises the following steps:
the first day:
(1) Adding 5ml YPD into a 50ml centrifuge tube, inoculating X-33 strain, and culturing at 30 ℃ overnight;
the next day:
(2) Transferring 50ul of the bacterial liquid to a 250ml conical flask of 50ml YPD after 10-12h, and culturing overnight until OD600=1.3-1.5;
and on the third day:
(3) Centrifuging at 4000rpm at 4 deg.C for 5min, resuspending with 10ml buffera, water bathing at 30 deg.C for 15min, and adding pre-cooled sterilized water to 50ml;
buffer A:20mlYPD +2ml2MHEPES (pH =8.0, filter sterilized) +0.5ml1MDTT (filter sterilized);
(4) Centrifuging at 4000rpm at 4 ℃ for 5min, and resuspending with 50ml of pre-cooled sterile water (containing 0.3ml of 2MHEPES, pH = 8.0);
(5) Centrifuging at 4000rpm at 4 ℃ for 5min and resuspending with 4ml of precooled 1 Msorbitol;
(6) Centrifugation is carried out at 4000rpm for 5min at 4 ℃, then 100ul of precooled 1Msorbitol is used for resuspension, the bacterial liquid is viscous at this time, 80 ul/tube split charging is carried out, and the mixture is placed on ice.
In this embodiment, preferably, yeast is electrotransferred via a linearized vector;
wherein, the yeast electrotransformation by the linearized vector comprises the following steps:
(1) Taking 80ul of competent cells, adding 6ug of linearized pPICZ alpha A-gene, uniformly mixing, and transferring to a precooled 0.2cm electric shock cup;
(2) Standing on ice for 5min;
(3) According to the yeast electric shock parameter setting (1.5kV, 25uF,200 omega), electric shock is carried out;
(4) 2ml of pre-cooled 1M sorbent + HEPES (10ml 1M sorbent +100ul 2M HEPES, pH = 8.0) was immediately added and transferred to a 2ml sterile centrifuge tube;
(5) Standing and incubating for 1-2h at 30 ℃;
(6) After dilution 5-fold, 10-fold, and 100-fold, 300 ul/plate was spread on 15cm YPD plates containing 100mg/l Zeocin, and cultured at 30 ℃ until colonies grew out.
In this embodiment, preferably, transformants are screened;
wherein, the screening of the transformant comprises the following steps:
(1) Randomly selecting 24 clones from the plate, and carrying out colony PCR; (colony PCR uses a rapid amplification yeast cloning kit which is independently developed by the company, and the product number is RY 8001);
(2) 2 of them were selected for sequencing, and 7 clones were retained after correct alignment for use.
In this example, preferably, the transformant expresses the lab;
wherein, the transformant expression small test comprises the following steps:
(1) Preparing 7 50ml conical flasks, respectively adding 5ml YPG medium, respectively inoculating the above clones verified to be correct, and culturing at 30 deg.C and 220rpm for 1-2 days until the bacterial liquid is saturated;
YPG medium: 1% yeast extract, 2% peptone, 1% glycerol;
(2) Transferring the mixture into a 50ml centrifuge tube, centrifuging the mixture for 5min at 4000rpm, and removing supernatant;
(3) Resuspending the cells in 5ml BMMY medium, transferring to a new sterile 50ml conical flask, adding methanol to a final concentration of 0.5%, and culturing at 28 ℃ and 220rpm for 6 days;
BMMY medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source, 100mMPBS buffer (pH = 5.7);
(4) Adding methanol (the final concentration is 0.5%) every 24 h;
(5) And collecting bacteria liquid in the morning of 6d, centrifuging at 4000rpm for 5min, and collecting supernatant for expression identification.
In this example, it is preferable that the transformant is expressed in an enlarged manner.
Wherein the method for expanding the expression of the transformant comprises the following steps:
(1) Adding 50ml YPG culture medium into a 250ml conical flask, inoculating an optimal expression transformant, and culturing at 30 ℃ and 220rpm for 1-2 days until the bacterium liquid is saturated;
(2) Transferring the mixture into a 50ml centrifuge tube, centrifuging the mixture for 5min at 4000rpm, and removing supernatant;
(3) Resuspending the cells in 50ml BMMY medium, transferring to a new sterile 250ml conical flask, adding methanol to a final concentration of 0.5%, and culturing at 28 ℃ and 220rpm for 6 days;
(4) Adding methanol (the final concentration is 0.5%) every 24 h;
(6) And collecting bacteria liquid in the morning of 6d, centrifuging at 4000rpm for 5min, and collecting supernatant for expression identification.
The above description is only for the purpose of illustrating the technical solutions of the present invention and not for the purpose of limiting the same, and other modifications or equivalent substitutions made by those skilled in the art to the technical solutions of the present invention should be covered within the scope of the claims of the present invention without departing from the spirit and scope of the technical solutions of the present invention.
Claims (9)
1. A yeast optimized process preparation method is characterized by comprising the following steps: comprises the steps of (a) preparing a mixture of a plurality of raw materials,
constructing a vector: predicting a target gene signal peptide, removing a signal peptide sequence, carrying out codon optimization according to a pichia pastoris expression system, and carrying out gene synthesis to pPICZ alpha A;
electrically transforming pichia pastoris into plasmid shape;
linearizing a target vector;
purifying and recovering the linearized vector;
preparing yeast electrotransformation competence;
yeast electrotransformation by linearized vector;
screening transformants;
a transformant expression small test;
transformants were expanded for expression.
2. The yeast optimized technological preparation method according to claim 1, characterized in that: predicting target gene signal peptide by using SignalP 4.0 and SignalP5.1;
when codon optimization is carried out on a pichia pastoris expression system, the SacI enzyme cutting site is avoided;
when the gene was synthesized to pPICZ. Alpha.A, the gene of interest was located next to the vector alpha-factor, with 6 XHis at the C-terminal band.
3. The yeast-optimized process preparation method according to claim 1, characterized in that: the linearization of the target vector comprises the following steps:
(1) Preparing a carrier enzyme digestion system according to the following table:
(2) Enzyme digestion is carried out at 37 ℃ overnight;
(3) Detecting by agarose gel electrophoresis, and taking plasmid which is not cut by enzyme as a reference;
(4) After enzyme digestion is detected successfully, inactivation is carried out for 20min at 65 ℃.
4. The yeast-optimized process preparation method according to claim 1, characterized in that: wherein, the linearized vector is purified and recovered; the method comprises the following steps:
(1) The carrier purification system was configured as follows:
(2) Standing at-20 deg.C for more than 35 min;
(3) Centrifuging at 12000rpm at 4 deg.C for 15min, and discarding the supernatant, wherein white precipitate on the wall can be observed;
(4) Adding 400ul of precooled 80% ethanol for resuspension and precipitation;
(5) Centrifuging at 12000rpm at 4 deg.C for 10min, discarding supernatant, and drying;
(6) The precipitate was dissolved by adding 10ul ddH 2O.
5. The yeast optimized technological preparation method according to claim 1, characterized in that: wherein, the preparation of the yeast electrotransformation competence comprises the following steps:
the first day:
(1) Adding 5ml YPD into a 50ml centrifuge tube, inoculating X-33 strain, and culturing at 30 ℃ overnight;
the next day:
(2) Transferring 50ul of the bacterial liquid to a 250ml conical flask of 50ml YPD after 10-12h, and culturing overnight until OD600=1.3-1.5;
and on the third day:
(3) Centrifuging at 4000rpm at 4 deg.C for 5min, resuspending with 10ml buffera, water bathing at 30 deg.C for 15min, and adding pre-cooled sterilized water to 50ml;
buffer A:20mlYPD +2ml2MHEPES (pH =8.0, filter sterilized) +0.5ml1MDTT (filter sterilized);
(4) Centrifuging at 4000rpm at 4 deg.C for 5min, and resuspending with 50ml of pre-cooled sterile water (containing 0.3ml of 2MHEPES, pH = 8.0);
(5) Centrifuging at 4000rpm at 4 ℃ for 5min and resuspending with 4ml of precooled 1 Msorbitol;
(6) Centrifugation is carried out at 4000rpm for 5min at 4 ℃, then 100ul of precooled 1Msorbitol is used for resuspension, the bacterial liquid is viscous at this time, 80 ul/tube split charging is carried out, and the mixture is placed on ice.
6. The yeast optimized technological preparation method according to claim 1, characterized in that: wherein, the yeast electrotransformation by the linearized vector comprises the following steps:
(1) Taking 80ul of competent cells, adding 6ug of linearized pPICZ alpha A-gene, uniformly mixing, and transferring to a precooled 0.2cm electric shock cup;
(2) Standing on ice for 5min;
(3) According to the yeast electric shock parameter setting (1.5kV, 25uF,200 omega), electric shock is carried out;
(4) 2ml of pre-cooled 1M sorbent + HEPES (10ml 1M sorbent +100ul 2M HEPES, pH = 8.0) was immediately added and transferred to a 2ml sterile centrifuge tube;
(5) Standing and incubating for 1-2h at 30 ℃;
(6) After dilution 5-fold, 10-fold, and 100-fold, 300 ul/plate was spread on 15cm YPD plates containing 100mg/l Zeocin, and cultured at 30 ℃ until colonies grew out.
7. The yeast optimized technological preparation method according to claim 1, characterized in that: wherein, the screening of the transformant comprises the following steps:
(1) Randomly selecting 24 clones from the plate, and carrying out colony PCR; (colony PCR uses a rapid amplification yeast cloning kit which is independently developed by the company, and the product number is RY 8001);
(2) 2 of them were selected for sequencing, and 7 clones were retained for use after correct alignment.
8. The yeast optimized technological preparation method according to claim 1, characterized in that: wherein, the transformant expression small test comprises the following steps:
(1) Preparing 7 50ml conical flasks, respectively adding 5ml YPG medium, respectively inoculating the above clones verified to be correct, and culturing at 30 deg.C and 220rpm for 1-2 days until the bacterial liquid is saturated;
YPG medium: 1% yeast extract, 2% peptone, 1% glycerol;
(2) Transferring the mixture into a 50ml centrifuge tube, centrifuging the mixture for 5min at 4000rpm, and removing supernatant;
(3) Resuspending the cells in 5ml BMMY medium, transferring to a new sterile 50ml conical flask, adding methanol to a final concentration of 0.5%, and culturing at 28 ℃ and 220rpm for 6 days;
BMMY medium: 1% yeast extract, 2% peptone, 1.34% yeast nitrogen source, 100mMPBS buffer (pH = 5.7);
(4) Adding methanol (the final concentration is 0.5%) every 24 h;
(5) And collecting bacteria liquid in the morning of 6d, centrifuging at 4000rpm for 5min, and collecting supernatant for expression identification.
9. The yeast optimized technological preparation method according to claim 1, characterized in that: wherein the method for expanding the expression of the transformant comprises the following steps:
(1) Adding 50ml of YPG culture medium into a 250ml conical flask, inoculating an optimal expression transformant, and culturing at 30 ℃ and 220rpm for 1-2 days until the strain liquid is saturated;
(2) Transferring the mixture into a 50ml centrifuge tube, centrifuging the mixture for 5min at 4000rpm, and removing supernatant;
(3) Resuspending the cells in 50ml BMMY medium, transferring to a new sterile 250ml conical flask, adding methanol to a final concentration of 0.5%, and culturing at 28 ℃ and 220rpm for 6 days;
(4) Adding methanol (final concentration is 0.5%) every 24 h;
(6) And collecting bacterial liquid in the morning at 6d, centrifuging at 4000rpm for 5min, and collecting supernatant for expression identification.
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| CN104946675A (en) * | 2015-07-02 | 2015-09-30 | 江苏敖众生物科技有限公司 | Cloning of egg white lysozyme gene (RJM) and yeast expression method thereof |
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