CN111411145A - Method for obtaining ribosome nascent-chain complex in yeast - Google Patents
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Abstract
The invention relates to a method for obtaining ribosome nascent-chain complex in yeast. The method comprises the following steps: (1) obtaining a yeast sample and carrying out pretreatment by using an cycloheximide solution; (2) resuspending the yeast sample subjected to the preliminary treatment in a buffer solution II and centrifuging to obtain a precipitate; (3) resuspending the pellet obtained in step (2) in yeast lysate and split into a first solution and a second solution; (4) extracting the total RAN from the first solution in step (3) and extracting the RNC from the second solution. By utilizing the method, the ribosome nascent-chain complex in the yeast can be completely obtained, and compared with the traditional extraction experimental method, the method overcomes the defects of complex extraction operation, low concentration, poor quality, more impurities and the like in the extraction of the yeast RNC.
Description
Technical Field
The invention relates to the technical field of sequencing of a translation group, in particular to a method for obtaining a ribosome nascent-chain complex in yeast.
Background
The expression system of Pichia pastoris (Pichia pastoris) is developed in recent years and is widely used for expressing a methanol nutrition type yeast expression system of foreign protein, the over-expression of the foreign protein in the Pichia pastoris is often accompanied with the change of the expression level of other genes, and the key problem of influencing the expression level of the foreign protein is conveniently found by analyzing the difference of the expression levels of the related genes.
However, compared with other cells, pichia pastoris has a firmer cell wall, and particularly pichia pastoris cells in a stationary phase have thicker cell wall and poor permeability, and the factors can influence the expression condition of gene proteins in cells in a cell removing method. The obstruction of the cell wall not only increases the difficulty of extracting RNA, but also the accuracy of subsequent gene expression level measurement is directly influenced by low-quality RNA.
The translatomics is an important subject of regulation in organisms newly discovered in recent years. According to the central principles, DNA is transcribed into mRNA, which is translated into protein polypeptides. During translation of mRNA according to codon information, ribosomes bound to mRNA migrate along the RNA chain and gradually bind to tRNA to form a protein polypeptide chain (Nascent peptide chain), i.e., Ribosome Nascent peptide-chain Complex (RNC). The traditional concept is that the gene is transcribed and translated, and the translatomics find that the transcription is not equal to the translation in the research process, and the translation is not carried out after partial gene is transcribed; even if the translation process is carried out, the translation ratio of different genes is different, the translation ratio of part of genes is high, and the translation ratio of part of genes is basically low; meanwhile, in the research process of the translational omics, the content of RNC-mRNA has an obvious relation with the corresponding protein expression rate in a sample, different translation rates determine the expression quantity of the protein, even the generation and the expression of newly developed protein, and only a complete RNC is extracted, so that the content of the RNC-mRNA which is translated is measured, the transcription and translation efficiency and different protein expressions are calculated by combining corresponding transcriptome and proteomic data, and the method has important significance for determining the cell phenotype, finding unknown single ratios and researching the proteomics.
At present, most RNC extraction utilizes a sucrose density gradient centrifugation method to carry out ultracentrifugation separation on a cell solution after membrane rupture, can extract RNC and quantify mRNA and a new peptide chain in the full length of ribosome, but the method only aims at animal fresh cells and has higher extraction efficiency. Before extracting fresh animal cells, a corresponding translation elongation inhibitor is added, and can enter the cells to fix ribosomes translating on mRNA so as to maintain the real condition during translation, and then high-quality RNC is extracted through serial operations.
Therefore, there is still a need in the art for a method for completely obtaining ribosome nascent-chain complex in fungal yeast (especially pichia pastoris), thereby facilitating research and application of protein transcription and translation.
Disclosure of Invention
Aiming at the defects in the prior art, the invention aims to provide a method for completely obtaining a ribosome nascent-chain complex in pichia pastoris, which overcomes the defects of complex operation, low concentration, poor quality, more impurities and the like in pichia pastoris RNC extraction compared with the traditional extraction experimental method.
The above purpose is realized by the following technical scheme of the invention:
in a first aspect, there is provided a method of obtaining ribosomal nascent peptide chain complexes in yeast, the method comprising the steps of: (1) obtaining a yeast sample and carrying out pretreatment by using an cycloheximide solution; (2) resuspending the yeast sample subjected to the preliminary treatment in a buffer solution II and centrifuging to obtain a precipitate; (3) resuspending the precipitate obtained in step (2) in yeast lysate and split-charging into a first solution and a second solution according to experimental requirements; (4) extracting the total RAN from the first solution in step (3) and extracting the RNC from the second solution. In a preferred embodiment, the detection object is pichia pastoris in fungal yeasts.
In the method according to the first aspect, pichia pastoris is pretreated by taking cycloheximide as an extension inhibitor in the early stage, RNC and mRNA which are translated in a sample are fixed, and the instant state in the translation process is kept; and then cracking the cell wall of the pichia pastoris by adopting a fungus lysate, crushing the cell, releasing substances in the pichia pastoris, performing gradient density centrifugation on the released substances by adopting a sucrose density centrifugation method according to different settleability of different substances, collecting the RNC in which the substances are completely precipitated, and detecting the integrity of the extracted ribosome nascent-chain complex by adopting a related detection method.
According to the method of the first aspect, in step (1), the pre-treatment comprises incubating the yeast sample in 50-200ug/m L of cycloheximide solution at room temperature for 2-10min, then incubating in buffer I for 5-20min and centrifuging to obtain the precipitate.
By utilizing the technical scheme, the cycloheximide can inhibit intracellular translation extension, and the expression state of instant translation is preserved, so that a similar fixing effect is achieved, and the ribosome nascent-chain complex in the yeast sample can be kept complete.
The method according to the first aspect, wherein in step (1), the buffer solution I is a solution containing 10-30mM potassium dihydrogen phosphate at pH6-8 and 5-20mM Dithiothreitol (DTT) and 50-200ug/m L cycloheximide.
The method according to the foregoing first aspect, wherein in step (2), the buffer solution II is potassium dihydrogen phosphate containing 10-30mM of pH6-8, 1-2M sorbitol, 0.2-1mM MgCl2100-200U/m L yeast lyase and 50-200ug/m L cycloheximide.
According to the method of the first aspect, in step (3), the yeast lysate is Tris-HC L containing 5-20mM pH7.4, 3-10mM MgCl250-150mM KCl, 1-3mM DTT, 50-150ug/m L cycloheximide and 0.5-2% Triton X-100 buffer solution.
By utilizing the technical scheme, different salt ions and Triton (which can be used as a detergent) are combined, and compared with the existing lysate, the lysate can effectively crack the cell wall of fungal yeast (such as pichia pastoris, especially in a stable period) with thicker cell wall and difficult cracking, so that a complete ribosome nascent peptide chain compound is obtained.
The method according to the first aspect, wherein in step (4), extracting total RNA comprises extracting total RNA using an extraction reagent. In a preferred embodiment, the extraction reagent is Trizol.
The method according to the foregoing first aspect, wherein in step (4), the extracting the RNC comprises the steps of: (a) centrifuging the second solution in a centrifuge at 0-4 deg.C to obtain supernatant and transferring to sucrose buffer solution for separation; (b) centrifuging in a 0-4 deg.C ultra-high speed centrifuge to obtain RNC in yeast sample; (c) extracting NAC-RNA from the RNC obtained in step (b) using RNC extraction reagents. In a preferred embodiment, the RNA extraction reagent is Trizol.
Through the technical scheme, the sucrose density gradient centrifugation is utilized, the sucrose solutions with different densities and the centrifugal force action can be combined, and the full separation effect is achieved according to the different sedimentation degrees of RNC and other substances.
The method according to the first aspect, wherein in the step (4), the sucrose buffer solution for separation is Tris-HC L containing 5-20mM of pH7.4, 3-10mM of MgCl250-200mM KCl, 1-3mM DTT and 50-200ug/m L cycloheximide buffer solution.
The method according to the first aspect, further comprising the step of snap freezing the pre-treated yeast sample with liquid nitrogen or a dry ice-ethanol system and storing and transporting the sample at a temperature of less than-80 ℃ after the step (1) and before the step (2). In a preferred embodiment, the temperature is preferably from-80 to-196 ℃.
In summary, the invention includes at least one of the following beneficial technical effects:
1. by utilizing the method, the ribosome nascent-chain complex in the yeast (especially pichia pastoris) sample can be completely obtained, so that the method can be used for analyzing different gene functions and carrying out combined analysis by combining other transcriptome data;
2. the lysis solution prepared by the invention has good effect on cell wall lysis and little influence on RNA, and an extension inhibitor of cycloheximide is added in the early stage, so that the translation is stopped immediately, the integrity can be ensured, the transportation and the storage are convenient, and the convenience is provided for further researching the yeast cell RNC.
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The invention will be further explained and explained with reference to the drawings. Those skilled in the art will appreciate that these drawings are provided solely for the purpose of enabling those skilled in the art to better understand the present invention and are not intended to limit the scope of the present invention in any way.
FIG. 1 is an electrophoresis chart of RNC-mRNA nucleic acids of Pichia samples collected at different growth stages from different strains isolated, where M: DNA marker; lanes 1-4 are: 113Y-1-RNC, 113Y-2-RNC, 113S-1-RNC and 113S-2-RNC;
FIG. 2 is a map of the RNC-mRNA nucleic acid Agilent2100 assay of Pichia samples collected at different growth stages from different strains isolated.
Detailed Description
The following examples further illustrate the embodiments of the present invention in detail.
At present, the RNC extraction method mainly utilizes a sucrose density gradient centrifugation method to carry out ultracentrifugation, and mainly aims at fresh cells of animals and plants, and the extraction difficulty of RNA and RNC of fungal yeast (particularly pichia pastoris) is increased due to the thick cell wall blocking effect of the fungal yeast, so that the effect of only utilizing the sucrose density gradient centrifugation method is not good. Moreover, because of the prevention effect of the thicker cell wall, the enzymolysis method cannot be adopted under the condition that RNA expression cannot be fixed in advance, because the method needs to be carried out under the condition of 37 ℃, the temperature can influence the expression transcription and translation conditions of certain genes to change, the problems of large sample consumption, more impurities, low purity and the like exist in the total RNA and RNC samples, and the method has an interference effect on the research of protein transcription and translation.
Based on this point, the inventors of the present application have conducted a great deal of research and obtained relatively good results, and have completed the present invention based on this research. Specifically, the inventors of the present application propose the following technical solutions: a method for obtaining a ribosome nascent peptide chain complex in yeast, the method comprising the steps of: (1) obtaining a yeast sample and carrying out pretreatment by using an cycloheximide solution; (2) resuspending the yeast sample subjected to the preliminary treatment in a buffer solution II and centrifuging to obtain a precipitate; (3) resuspending the pellet obtained in step (2) in yeast lysate and split into a first solution and a second solution; (4) extracting the total RAN from the first solution in step (3) and extracting the RNC from the second solution.
The present invention will be described in further detail with reference to specific examples.
Implement one
A. Pretreatment of pichia pastoris samples in different growth periods
The method comprises the following steps of (1) preprocessing the pichia pastoris samples in different growth periods, wherein the preparation process comprises the following steps:
the method comprises the following steps: preparation of solutions
1) Cycloheximide mother liquor with a concentration of 10mg/m L (Sigma-Aldrich).
2) Buffer I20 mM, pH7.4 potassium dihydrogen phosphate, 10mM Dithiothreitol (DTT), 100ug/m L cycloheximide solution, sterile rnase-free water and sterile instruments were used for preparation, and the solution was sterilized by filtration.
3) Liquid nitrogen or dry ice-ethanol
Step two: pretreatment of a pichia pastoris sample:
1) separately collecting the cultured Pichia pastoris bacterial liquid of different periods, about 1 × 107113Y (72 h and 96 h) and 113S (72 h and 96 h) cells, collected after centrifugation in sterilized 1.5m L EP tubes for pre-treatment;
2) adding a proper amount of 100ug/m L cycloheximide solution into the collected Pichia pastoris sample, immersing the sample precipitate, resuspending, and incubating at room temperature for 5 min;
3) buffer I was added to the resuspension solution and incubated at 30 ℃ for 10 min.
4) Placing the cracked sample in a centrifuge precooled to 0-4 ℃ and centrifuging for 3min at the rotating speed of 3000 × g, and carefully sucking and removing the supernatant by using a gun head;
5) and soaking the residual precipitate sample into liquid nitrogen with a proper volume, and quickly freezing by adopting a liquid nitrogen or dry ice-ethanol system, wherein the residual precipitate sample can be used for storage or transportation.
B. Extraction and detection of RNC (radio network controller) of pichia pastoris samples in different growth periods
The method comprises the following steps: preparation of solutions
1) Cycloheximide mother liquor with concentration of 10mg/m L (Sigma-Aldrich);
2) buffer II 20mM potassium dihydrogen phosphate pH7.4, 1.2M sorbitol, 0.5mM MgCl2150U/m L yeast lyase, 100ug/m L cycloheximide, sterile water and sterile instruments are used during preparation, and the solution is filtered and sterilized after the preparation;
3) ribosome buffer solution 10mM, Tris-HC L of pH7.4, 5mM MgCl2100mM KCl, 2mM DTT, 100ug/m L cycloheximide, sterile water without RNase and sterile instruments are used for preparation, and the prepared solution is filtered and sterilized;
4) pichia pastoris lysate: dissolving Triton X-100 in the ribosome buffer solution, wherein the final concentration of the Triton X-100 is 1 percent by volume;
5) sucrose solution for separation: sucrose was dissolved in the above ribosome buffer solution at a final sucrose concentration of 30% (W/V) and pre-cooled to 0-6 ℃.
Step two: pretreatment of a pichia pastoris sample:
1) resuspending the pre-treated sample in the early stage by using a buffer solution II, and incubating for 10min on ice;
2) placing the cracked sample in a centrifuge precooled to 0-4 ℃ and centrifuging for 3min at the rotating speed of 3000 × g, and carefully sucking and removing the supernatant by using a gun head;
3) adding 1ml of Pichia pastoris lysate for heavy suspension, and carrying out ice bath for 10 min;
4) wherein all samples are divided into two parts, one part is used for extracting total RNA, and the other part is used for extracting RNC; extracting total RNA samples by adopting an RNA extraction reagent Trizol and extracting the total RNA according to a method of an instruction manual;
5) after the RNC sample is extracted and incubated, placing the cracked sample in a centrifuge which is precooled to 0-4 ℃ and centrifuging for 10min at the rotating speed of 20000 × g, carefully sucking the supernatant by a gun head, and transferring the supernatant to a sucrose buffer solution for separation which is precooled in advance and is 12.5m L;
6) placing the transferred sucrose buffer solution for separation in a super-high speed centrifuge which is cooled to 0-4 ℃, and centrifuging at the rotating speed of 185000 × g for 5h to obtain RNC in different pichia pastoris samples;
7) RNC-RNA was extracted from RNC using Trizol (Invitrogen) which is an RNA extraction reagent according to the manual method.
In order to detect the method, undegraded RNC-RNA can be extracted from different growth periods of pichia pastoris, 2 percent agarose is adopted to carry out electrophoretic separation on the RNC-RNA in the extracted pichia pastoris sample, SybrGreen II is used for dyeing, and an ultraviolet gel imaging system is used for imaging detection; and the results were also tested using Agilent 2100.
The results of the RNC-mRNA extraction concentrations of pichia samples collected from different fungi at different growth times are shown in table 1 below. As a result, as shown in Table 1, the RNA purity (expressed as OD (260/280)) of all samples was 1.8-2.0, and thus the RNA extraction quality was good. In addition, the RNC-mRNA extraction concentration is combined, so that the RNC-mRNA extraction effect is good, and the RNA high quality standard is met.
TABLE 1 concentration of RNC-mRNA extracts from Pichia samples collected from different fungi at different growth times
The results of RNC-mRNA nucleic acid electrophoresis of pichia pastoris samples collected from different fungi at different growth times and RNC-mRNA nucleic acid Agilent2100 detection of pichia pastoris samples collected from different fungi at different growth times are shown in fig. 1 and 2, respectively. As shown in the figures 1 and 2, after electrophoresis and Agilent2100 detection of the extracted RNA, bands of all samples at 28S, 18S and 5S are obvious and pollution-free, and the results prove that the extracted Pichia pastoris RNC-RNA has the advantages of high quantity, clear band type, no impurity band and no degradation, so that the method can be used for completely obtaining the Pichia pastoris RNC-mRNA.
The embodiments of the present invention are merely preferred embodiments, and not intended to limit the scope of the present invention, and all equivalent changes made according to the spirit and principles of the present invention should be covered by the present invention.
Claims (10)
1. A method for obtaining a ribosome nascent peptide chain complex in yeast, comprising the steps of:
(1) obtaining a yeast sample and carrying out pretreatment by using an cycloheximide solution;
(2) resuspending the yeast sample subjected to the preliminary treatment in a buffer solution II and centrifuging to obtain a precipitate;
(3) resuspending the pellet obtained in step (2) in yeast lysate and split into a first solution and a second solution;
(4) extracting the total RAN from the first solution in step (3) and extracting the RNC from the second solution.
2. The method of claim 1, wherein the yeast is pichia pastoris.
3. The method according to claim 1 or 2, wherein in step (1), the pre-treatment comprises incubating the yeast sample in 50-200ug/m L of cycloheximide solution at room temperature for 2-10min, then incubating in buffer I for 5-20min and centrifuging to obtain the precipitate.
4. The method according to claim 1 or 2, wherein in step (1), the buffer solution I is a solution containing 10-30mM potassium dihydrogen phosphate (koh) at pH6-8 and 5-20mM Dithiothreitol (DTT) and 50-200ug/m L cycloheximide.
5. The method according to claim 1 or 2, wherein in step (2), the buffer solution II is a solution containing 10-30mM potassium dihydrogen phosphate at pH6-8, 1-2M sorbitol, 0.2-1mM MgCl2, 100-200U/M L yeast lyase, 50-200ug/M L cycloheximide.
6. The method according to claim 1 or 2, wherein in step (3), the yeast lysate is Tris-HC L containing 5-20mM of pH7.4, 3-10mM of MgCl250-150mM KCl, 1-3mM DTT, 50-150ug/m L cycloheximide and 0.5-2% Triton X-100 buffer solution.
7. The method of claim 1 or 2, wherein in step (4), extracting total RNA comprises extracting total RNA using an extraction reagent.
8. The method of claim 1 or 2, wherein in step (4), extracting the RNC comprises the steps of: (a) centrifuging the second solution in a centrifuge at 0-4 deg.C to obtain supernatant and transferring to sucrose buffer solution for separation; (b) centrifuging in a 0-4 deg.C ultra-high speed centrifuge to obtain RNC in yeast sample; (c) extracting RNC-RNA from the RNC obtained in step (b) using RNC extraction reagent.
9. The method according to claim 1 or 2, wherein in step (4), the sucrose buffer solution for separation is Tris-HC L containing 5-20mM of pH7.4, 3-10mM of MgCl250-200mM KCl, 1-3mM DTT and 50-200ug/m L cycloheximide buffer solution.
10. The method according to claim 1 or 2, wherein the method further comprises the steps of quick freezing the yeast sample subjected to the preliminary treatment with liquid nitrogen or a dry ice-ethanol system, and storing and transporting the yeast sample at a temperature of less than-80 ℃ after the step (1) and before the step (2).
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Citations (4)
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| CN104186460A (en) * | 2014-08-07 | 2014-12-10 | 暨南大学 | Method for complete cryopreservation and thawing of intracellular RNC (ribosome nascent-chain complex) |
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| CN104186460A (en) * | 2014-08-07 | 2014-12-10 | 暨南大学 | Method for complete cryopreservation and thawing of intracellular RNC (ribosome nascent-chain complex) |
| CN104262478A (en) * | 2014-09-05 | 2015-01-07 | 暨南大学 | Method for fully obtaining interstitial ribosome nascent-chain complex |
| CN104961813A (en) * | 2015-06-16 | 2015-10-07 | 暨南大学 | Method of completely acquiring ribosome nascent-chain complex from plant tissues and application of method |
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Application publication date: 20200714 |