CN109060744B - Fluorescence analysis method for secretion expression quantity of pichia pastoris recombinant protein - Google Patents

Fluorescence analysis method for secretion expression quantity of pichia pastoris recombinant protein Download PDF

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CN109060744B
CN109060744B CN201810922217.6A CN201810922217A CN109060744B CN 109060744 B CN109060744 B CN 109060744B CN 201810922217 A CN201810922217 A CN 201810922217A CN 109060744 B CN109060744 B CN 109060744B
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pichia pastoris
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CN109060744A (en
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林影
梁书利
李承�
袁清焱
韩双艳
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South China University of Technology SCUT
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    • G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
    • G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64—Fluorescence; Phosphorescence
    • G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
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    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/43504—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates
    • C07K14/43595—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from invertebrates from coelenteratae, e.g. medusae
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Abstract

The invention discloses a fluorescence analysis method for secretion expression quantity of recombinant pichia pastoris protein, and relates to the technical field of genetic engineering. The invention discloses a pichia pastoris endoplasmic reticulum protein fusion expression fluorescent protein, and the secretory expression quantity of a recombinant protein is represented by detecting the fluorescence value of thalli. The method can rapidly analyze the secretion expression quantity of the recombinant protein in the pichia pastoris by using the fluorescence value of the fluorescent protein (EGFP), does not need complicated operation, is simple, convenient and rapid, is suitable for detecting the secretion expression quantity of the recombinant protein in biological engineering and genetic engineering, and is favorable for rapidly detecting the recombinant strain of the high-efficiency secretion expression recombinant protein in the pichia pastoris.

Description

Fluorescence analysis method for secretion expression quantity of pichia pastoris recombinant protein
Technical Field
The invention relates to the technical field of genetic engineering, relates to a fluorescence analysis method for secretion expression quantity of pichia pastoris recombinant protein, and particularly relates to a fluorescence analysis method for secretion expression quantity of pichia pastoris recombinant protein based on endoplasmic reticulum membrane protein Sec 63.
Background
Yeast has advantages of efficiently performing complex post-translational modification and secreting recombinant proteins having a biological functional form to the outside of cells, and is widely used for producing recombinant proteins for industrial applications. Wherein, the Pichia pastoris has the advantages of having a strong promoter P strictly regulated and controlled by methanolAOX1Proper glycosylation, simple culture, easy realization of high-density fermentation, easy purification of secretory expression recombinant protein and the like, and at present, more than 5000 recombinant proteins are successfully expressed in pichia pastoris.
Although the secretion expression level of most recombinant proteins can reach the level of gram per liter, the secretion expression level of many recombinant proteins is low, and recombinant strains need to be modified to improve the secretion expression level of the recombinant proteins, such as increasing gene expression cassette copies, improving protein folding flux and the like. However, the modified strain still needs to detect the secretion expression amount of the recombinant protein to verify whether the modification is effective, and then on the basis of the obtained recombinant strain, the next modification is carried out to further improve the secretion expression amount of the recombinant protein. Methods for detecting the expression level of different recombinant proteins are different, for example, the enzyme activity of recombinant enzymes is generally detected, and the processes generally require a certain reaction time and are relatively complicated. At present, no better method is available for rapidly analyzing the secretion expression quantity of the recombinant protein. Therefore, it is important to find a method for rapidly analyzing the secretory expression level of a recombinant protein.
Disclosure of Invention
In order to overcome the defects of the prior art, the invention aims to provide a fluorescence analysis method for the secretion expression quantity of the recombinant pichia pastoris protein, which has the characteristics of simplicity, convenience and rapidness and is suitable for detecting the secretion expression quantity of the recombinant protein in biological engineering and genetic engineering.
The purpose of the invention is realized by the following technical scheme:
the invention provides an application of a pichia pastoris strain containing endoplasmic reticulum membrane protein fusion expression fluorescent protein in detecting the secretion expression quantity of pichia pastoris recombinant protein.
The detection of the secretion expression quantity of the recombinant pichia pastoris protein is realized by detecting the fluorescence value of the thallus.
The invention provides a fluorescence analysis method for secretion expression quantity of recombinant pichia pastoris protein, which comprises the following steps:
the pichia pastoris endoplasmic reticulum protein is fused to express the fluorescent protein, and the secretion expression quantity of the recombinant protein is represented by detecting the fluorescence value of the thallus;
the method specifically comprises the following steps:
(1) constructing a gene segment of endoplasmic reticulum membrane protein-fluorescent protein, and transforming the gene segment into a competent cell of pichia pastoris;
(2) preparing pichia pastoris competent cells of endoplasmic reticulum membrane protein fusion expression fluorescent protein;
(3) transforming a vector for expressing the recombinant protein into the competent cells prepared in the step (2), and coating the competent cells on corresponding screening plates to obtain positive transformants;
(4) inoculating the positive transformant into a fermentation culture medium for fermentation culture, detecting the fluorescence value of thalli of the transformant, and representing the secretion expression quantity of the recombinant protein.
Preferably, the endoplasmic reticulum membrane protein is Sec 63.
Preferably, the fluorescent protein is enhanced green fluorescent protein EGFP.
Preferably, the Pichia pastoris is Pichia pastoris (GS 115).
The fluorescence analysis method for the secretion expression quantity of the pichia pastoris recombinant protein is applied to the aspect of detecting the secretion expression quantity of the pichia pastoris recombinant protein.
The detection of the secretion expression quantity of the recombinant pichia pastoris protein is realized by detecting the fluorescence value of the thallus.
Preferably, the embodiment of the invention takes the endoplasmic reticulum protein Sec63, the enhanced green fluorescent protein EGFP and the Pichia pastoris GS115 as examples:
the method specifically comprises the following steps of expressing enhanced green fluorescent protein EGFP by fusion of endogenous Sec63 protein of pichia pastoris, detecting the EGFP fluorescence value and the recombinant protein secretion expression quantity when recombinant protein is secreted and expressed by using the strain, and analyzing the relationship between the recombinant protein secretion expression quantity and the EGFP fluorescence value:
(1) preparing an SEC63-EGFP fragment by using overlap extension PCR, and transforming the fragment into pichia pastoris GS115 to obtain Sec63 fusion expression EGFP recombinant strain GS 115-E;
(2) preparing a competent cell of GS115-E, linearizing a vector carrying six copies of phytase gene derived from Citrobacter (Citrobacter aromaticus) CGMCC1696, transforming the vector into the competent cell, detecting the enzyme activity of the cell EGFP and the enzyme activity of the phytase during induction, and analyzing the correlation between the enzyme activity of the phytase and the enzyme activity of the cell EGFP.
(3) After being linearized, vectors respectively carrying one, two and four copies of phytase genes are transformed into competent cells of GS115-E, the bacterial EGFP fluorescence value and the phytase enzyme activity of each recombinant strain are detected when the recombinant strains are induced for 96 hours, and the correlation between the phytase enzyme activity and the bacterial EGFP fluorescence value is analyzed.
(4) Preparing the recombinant strain competent cells expressing the phytase obtained in the step (2), linearizing a vector carrying unfolded protein response transformation factor genes, then transforming the vector into the competent cells, detecting the EGFP fluorescence value and the phytase enzyme activity between the two strains, and analyzing the difference between the increase of the EGFP fluorescence value and the increase of the phytase enzyme activity between the two strains.
(5) Linearizing a vector carrying xylanase gene derived from Bacillus halodurans (Bacillus halodurans) C125, converting the vector into competent cells of GS115-E, detecting the EGFP fluorescence value of the strain and the xylanase enzyme activity during induction, and analyzing the correlation between the xylanase enzyme activity and the EGFP fluorescence value of the strain.
Compared with the prior art, the invention has the following advantages and effects:
the EGFP fluorescence value is used in the invention, the secretion expression quantity of the recombinant protein in the pichia pastoris can be rapidly analyzed, the complex operation is not needed, and the rapid detection of the recombinant strain with the efficient secretion expression of the recombinant protein in the pichia pastoris is facilitated.
Drawings
FIG. 1 is a cell growth assay for Sec63 fusion-expressing EGFP strain GS 115-E; wherein (a) the strain GS115-E induces a fluorescence microscopic observation result for 96 hours, wherein a solid arrow represents a nuclear membrane layer of endoplasmic reticulum, and a dotted arrow represents a cortical layer of the endoplasmic reticulum; (b) growth curves of the strains GS115 and GS 115-E.
FIG. 2 shows the detection of phytase activity and fluorescence value of the recombinant strain GS115-E/P6c obtained when the strain GS115-E is used to express phytase; wherein, (a) phytase enzyme activity and thallus EGFP fluorescence curves of the strain GS115-E/P6c, wherein a solid graph represents phytase enzyme activity, and a hollow graph represents thallus EGFP fluorescence value; (b) and (3) a correlation graph of the phytase enzyme activity value and the bacterial EGFP fluorescence value of the strain GS115-E/P6 c.
FIG. 3 is a graph showing the correlation between phytase activity and the fluorescence value of the strain at 96h induction time of strains containing different phytase gene expression cassette copy numbers.
FIG. 4 is a graph of the response of the transcription factor HAC1 using co-expressed unfolded proteiniThe phytase enzyme activity and the thallus fluorescence value of the recombinant strain H/P6c obtained when the phytase expression quantity is further increased are detected; wherein, (a) the phytase activity and the thallus EGFP fluorescence of the strain H/P6c are induced for 96 hoursA light value; (b) and (3) a correlation graph of the phytase enzyme activity value and the bacterial EGFP fluorescence value during the induction period of the strain H/P6 c.
FIG. 5 shows the xylanase enzyme activity and the bacterial fluorescence value detection of the recombinant strain GS115-E/X4c obtained when xylanase is expressed by using the strain GS 115-E; wherein, (a) xylanase enzyme activity and thallus EGFP fluorescence curve of a strain GS115-E/X4 c; (b) and (3) a correlation graph of the xylanase enzyme activity value and the bacterial EGFP fluorescence value of the strain GS115-E/X4 c.
FIG. 6 is a schematic diagram of the present invention for characterizing the secretion expression amount of recombinant protein by detecting EGFP fluorescence value.
Detailed Description
The present invention will be described in further detail with reference to examples and drawings, but the present invention is not limited thereto.
The invention discloses a schematic diagram for representing the secretion expression amount of a recombinant protein by detecting an EGFP fluorescence value, which is shown in figure 6.
The examples relate to the following biomaterials:
plasmid pPICZA-EGFP was described in DOI: 10.1007/s10529-012 and 1055-8'.
Plasmid pPICZA-CREG357CIs disclosed in the document "DOI: 10.1038/s 41598-017-11494-5".
Plasmid pPICZA- α E10-HKA/(Phy)6、pPICZA-αE10-phy-HKA、pPICZA-αE10-HKA/(Phy)2、pPICZA-αE10-HKA/(Phy)4pPICZA-HAC1 is disclosed in DOI 10.1186/s 12896-015-0204-2.
Plasmid pHKa-4 Xyn10 is disclosed in the literature "Lin Xiao Qiong. protein group research of high-efficiency expression xylanase recombinant Pichia pastoris cells based on iTRAQ technology [ D ]. southern China university, 2013".
Example 1 construction of Sec63 fusion expression enhanced Green fluorescent protein host strains
(1) The upstream and downstream portions of the SEC63 gene (GenBank accession XP-002493824.1) were amplified using the Pichia pastoris GS115 genome as a template and primers P1 and P2 and P3 and P4. Plasmid pPICZA-EGFP (disclosed in the reference "DOI: 10.1007/s 10529-012-" 1055-8 ") was used as a templatePlate, primer P5 and P6 were used to amplify the gene fragment of the enhanced green fluorescent protein EGFP. With plasmid pPICZA-CREG357C(disclosed in the reference "DOI: 10.1038/s 41598-017-11494-5") as a template, a CRE expression cassette fragment was amplified using primers P7 and P8. Four PCR products obtained after recovery and purification of the gel are used as templates, and primers P1 and P4 are used for amplifying a fragment SEC 63-EGFP.
P1：5′-GCTAATAAATGGGCTGGTCGAT-3′；
P2：5′-TTTATCTTCACCCTCATCTTCC-3′；
P3：5′-TACATTATACGAACGGTAGTGATAGTGACAGATCAAA-3′；
P4：5′-GGAGGGAAGATTCACTCCAAAA-3′；
P5：5′-GAAGATGAGGGTGAAGATAAAGTGAGCAAGGGCGAGGAGCT-3′
P6：5′-AGATCTATAACTTCGTATAATGTATGCTATACGAACGGTATTACTTGTACAGCTCGTCCA-3′；
P7：5′-TACCGTTCGTATAGCATACATTATACGAAGTTATAGATCTAACATCCAAAGACG-3′；
P8：5′-TACCGTTCGTATAATGTATGCTATACGAAGTTATGTCTCCAGCTTGCAAATTAA-3′；
(2) The SEC63-EGFP fragment gel was recovered and purified, and then transformed into competent cells of Pichia pastoris GS115 (purchased from Invitrogen, Life technologies, Inc., USA) and screened for positive transformants on YPDZ bleomycin resistant plates.
(3) The correctly identified transformants were selected and inoculated into YPM medium and cultured at 30 ℃ and 250rpm for 24 h. mu.L of the medium was streaked onto YPD plates and cultured at 30 ℃ and 250rpm for 36 hours. The obtained transformant was named GS 115-E.
(4) The recombinant strain GS115-E and the wild pichia pastoris GS115 are respectively inoculated to a BMGY culture medium at the same time, and cultured for 20h at 30 ℃ and 250 rpm. The cells were harvested by centrifugation at 6000rpm for 5min at 4 ℃ and then resuspended in BMMY medium to the starting OD600Approximately 0.5, 30 ℃, 250rpm shake culture, daily supplemented with 1% final concentration methanol and sampled. Measurement of cell OD600And cells were observed using a confocal laser microscope.
Cells of strain GS115-E were observed by confocal laser microscopy at 96h in methanol culture and the endoplasmic reticulum nuclear envelope of these cells was found to have a continuous fluorescent appearance and the cortical layer to have an interrupted fluorescent appearance (FIG. 1-a), indicating that Sec63 has successfully fused to express EGFP. The growth curves of strain GS115-E were similar to that of GS115 during methanol culture (FIG. 1-b), indicating that Sec63 fusion does not affect cell growth after expression of EGFP.
Example 2 relationship between the expression level of recombinant Phytase and the fluorescence level of EGFP in the same Strain at different Induction times
(1) Preparation of Pichia pastoris GS115-E competent cell
a. Inoculating Pichia pastoris strain into 100mL YPD, culturing at 30 deg.C and 200rpm to OD600＝1.5±0.3；
b. Subpackaging into 250 mL centrifuge tubes, centrifuging at room temperature at 1500g for 5 min;
c. discard the supernatant, add 40mL of fresh LDST solution [ 100mM LiAc,10mM dithioritol, 0.6 Msabinol and10mM Tris-HCl, pH 7.5 ] (25 ℃), and incubate for 30min at 30 ℃;
d. centrifuge at 1500g for 5min at room temperature. Discarding the supernatant, resuspending the thallus with 1mL of 1M sorbitol in an ice bath, and transferring to a 1.5mL Ep tube;
e. the thalli is washed 3 times by 1mL ice bath 1M sorbierite;
f. finally, the cells were resuspended in 400. mu.L ice bath 1M sorbitol and aliquoted into 80. mu.L/tube. Used immediately or stored frozen at-80 ℃.
(2) Plasmid pPICZA- α E10-HKA/(Phy) carrying six copies of phytase gene PHY (GenBank: ABI98040.1) derived from Citrobacter (Citrobacter ammoniagenes) CGMCC16966(published in the reference "DOI: 10.1186/s 12896-015-.
(3) The resulting GS115-E/P6c and plasmid pPICZA- α E10-HKA/(Phy)6The recombinant strain GS115/P6c obtained by transforming into pichia pastoris GS115 competent cells is respectively inoculated to BMGY culture medium at the same time, and the BMGY culture medium is cultured for 20h at 30 ℃ and 250 rpm. Centrifuging at 6000rpm and 4 deg.C for 5min to collect cells,the collected cells were then resuspended in BMMY medium to the starting OD600Approximately 0.5, 30 ℃, 250rpm shake culture, daily supplemented with 1% final concentration methanol and sampled. The sample was centrifuged at 6000rpm at 4 ℃ for 5min to separate the biomass and the fermentation supernatant. And detecting enzyme activity of the bacterial EGFP fluorescence and fermentation supernatant phytase.
a. Determination of bacterial EGFP fluorescence:
after washing the collected cells three times with 200. mu.L of a resuspension buffer, the cells were resuspended in 200. mu.L of a resuspension buffer and the fluorescence intensity was measured on a microplate reader (excitation wavelength 488nm, emission wavelength 520 nm).
b. The method for measuring the enzyme activity of the phytase comprises the following steps:
adding appropriate amount of sodium acetate buffer (0.25mol/L, pH 5.50) into the supernatant of the fermentation liquid, diluting by appropriate times to a final volume of 1mL, and preheating at 37 deg.C for 5 min. 2mL of sodium phytate solution (0.00761mol/L, pH 5.50) was added, mixed, and reacted at 37 ℃ for 30 min. 2mL of a freshly prepared color developing/stopping solution (2.35g/L ammonium vanadate solution: 100g/L ammonium molybdate solution: 30% nitric acid: 1:2) was added thereto, and after standing at room temperature for 10min, the absorbance at 415nm was measured.
Definition of phytase activity: at 37 ℃, under the condition of pH value of 5.5, the enzyme amount of 1 mu mol of inorganic phosphorus released by hydrolyzing the sodium phytate solution per minute is l enzyme activity unit (U).
The calculation formula of the phytase activity is as follows: enzyme activity (U/mL) ═ nxVt×A/(ε×30×Ve/103) (ii) a n: dilution times of enzyme liquid; a: a change value of absorbance; vt: the total volume of the enzyme reaction solution; ve: volume of enzyme solution in the enzyme reaction solution; epsilon: molar absorptivity.
The enzyme production curves of GS115-E/P6c and GS115/P6c are similar during methanol induction (FIG. 2-a), and the experimental result shows that the fusion expression of EGFP on the basis of Sec63 has no influence on the expression amount of the recombinant protein. In addition, bacterial EGFP fluorescence values of the strain GS115-E/P6c during induction gradually increased with the increase of induction time, and the curve is similar to the phytase activity curve (FIG. 2-a). Correlation analysis is carried out on the fluorescence value of the EGFP of the thalli and the enzyme activity value of the phytase of the fermentation supernatant, and the correlation coefficient R between the fluorescence value of the EGFP of the thalli and the enzyme activity value of the phytase of the fermentation supernatant is found20.9807 (FIG. 2-b) The relationship between the two is proved to be highly relevant (0.8 < | R | < 1), and the fluorescence quantity of Sec63-EGFP can be used for characterizing the secretion expression quantity of phytase. The determination of the secretory expression quantity of the phytase requires about 60min, while the detection of the fluorescence quantity of the bacterial EGFP only requires about 6min, and the secretory expression quantity of the recombinant phytase can be rapidly detected by using a fluorescence analysis method based on Sec 63.
Example 3 relationship between the secretion expression level of recombinant Phytase and the fluorescence level of EGFP in different strains at the same induction time
(1) Plasmids pPICZA- α E10-PHY-HKA, pPICZA- α E10-HKA/(PHY) carrying one, two, four copies of phytase gene PHY, respectively2、pPICZA-αE10-HKA/(Phy)4(published in the reference "DOI: 10.1186/s 12896-015-.
(2) GS115-E/P1c, GS115-E/P2c, GS115-E/P4c and GS115-E/P6c were simultaneously inoculated into BMGY medium, respectively, and cultured at 30 ℃ and 250rpm for 20 hours. The cells were harvested by centrifugation at 6000rpm for 5min at 4 ℃ and then resuspended in BMMY medium to the starting OD600Approximately 0.5, 30 ℃, 250rpm shake culture, daily supplemented with 1% final concentration methanol and sampled. The sample was centrifuged at 6000rpm at 4 ℃ for 5min to separate the biomass and the fermentation supernatant. And detecting the EGFP fluorescence value of the thalli and the phytase activity of fermentation supernatant.
When the strain is induced by methanol for 96 hours, the phytase secretion expression amount of the strain gradually increases along with the increase of the copy number (figure 3). The correlation between the phytase secretion expression amount of each strain and the EGFP fluorescence amount of the strain is analyzed, and the correlation coefficient R between the phytase secretion expression amount and the EGFP fluorescence amount is found20.9677 (FIG. 3), which shows that the relationship between the two is highly correlated (0.8 < | R | < 1), further indicating that the fluorescence quantity of Sec63-EGFP can be used to characterize the secretion expression quantity of phytase.
Example 4 relationship between the secretion expression level of recombinant Phytase and the fluorescence level of EGFP when the expression level of Phytase is further increased
(1) Preparation of recombinantStrain GS115-E/P6c competent cells and will carry unfolded protein responsive transcription factor gene HAC1iThe plasmid pPICZA-HAC1 (disclosed in the reference "DOI: 10.1186/s 12896-015-0204-2") used MSsI to linearize GS115-E/P6c competent cells and screened for positive transformants on YPDZ bleomycin resistant plates, and the resulting positive rotor was designated H/P6 c.
(2) GS115-E/P6c and H/P6c were simultaneously inoculated into BMGY medium, respectively, and cultured at 30 ℃ and 250rpm for 20 hours. The cells were harvested by centrifugation at 6000rpm for 5min at 4 ℃ and then resuspended in BMMY medium to the starting OD600Approximately 0.5, 30 ℃, 250rpm shake culture, daily supplemented with 1% final concentration methanol and sampled. The sample was centrifuged at 6000rpm at 4 ℃ for 5min to separate the biomass and the fermentation supernatant. And detecting the enzyme activity of the phytase of the thallus fluorescence and the fermentation supernatant.
When the strain is induced for 96 hours, the EGFP fluorescence value of the thallus of the strain H/P6c is improved by 38 percent compared with GS115-E/P6c (figure 4-a), meanwhile, the phytase enzyme activity of the fermentation supernatant of the strain H/P6c is improved by 40 percent compared with GS115-E/P6c (figure 4-a), and the results of the two are similar (P6/GS 6/S, respectively)<0.05), and further indicates that the fluorescence quantity of Sec63-EGFP can be used for characterizing the secretory expression quantity of phytase. In addition, the correlation analysis is carried out on the EGFP fluorescence value of the thallus of the strain H/P6c and the enzyme activity value of the phytase of the fermentation supernatant, and the correlation coefficient R between the two values is found20.9554 (FIG. 4-b), which shows that the relationship between the two is highly correlated (0.8 < | R | < 1), and further shows that the fluorescence quantity of Sec63-EGFP can be used to characterize the secretion expression quantity of phytase.
Example 5 relationship between the secretion expression level of recombinant xylanase and the EGFP fluorescence level of strains expressing xylanase at different induction times
(1) A plasmid pHKa-4 Xyn10 (disclosed in the reference "Lin Xiao Qiong. proteomics research of recombinant Pichia pastoris cells for high-efficiency expression xylanase based on the iTRAQ technology [ D ]. southern China university, 2013") carrying a xylanase Gene XYN (NCBI Gene ID:891394) derived from Bacillus halodurans C125 is linearized with Kpn2I, then electrically transformed into Pichia pastoris GS115-E competent cells, and positive transformants are screened on a histidine auxotrophic plate, and the obtained positive transformant is named as GS 115-E/X4C.
(2) GS115-E/X4c was inoculated into BMGY medium and cultured at 30 ℃ and 250rpm for 20 hours. The cells were harvested by centrifugation at 6000rpm for 5min at 4 ℃ and then resuspended in BMMY medium to the starting OD600Approximately 0.5, 30 ℃, 250rpm shake culture, daily supplemented with 1% final concentration methanol and sampled. The sample was centrifuged at 6000rpm at 4 ℃ for 5min to separate the biomass and the fermentation supernatant. And detecting the activity of the bacteria fluorescence and the xylanase of the fermentation supernatant.
The xylanase activity determination method comprises the following steps:
adding appropriate amount of glycine buffer solution (0.5mol/L, pH 9.0) into the supernatant of the fermentation liquid, diluting by appropriate times to a final volume of 1mL, transferring the reaction liquid into a 25mL colorimetric tube, adding 1mL of 10mg/mL xylan solution, mixing, and reacting at 70 ℃ for 30 min. Immediately adding 3mL of 3, 5-dinitrosalicylic acid solution, gently mixing, boiling in a water bath for 10min, adding distilled water to constant volume of 25mL, and taking the reaction solution to measure the absorption value at a wavelength of 540 nm.
Xylanase activity definition: the enzyme amount which releases 1. mu. mol of reducing sugar by hydrolyzing 10mg/mL of xylan solution per minute at 70 ℃ under a pH of 9.0 is one enzyme activity unit (U).
Xylanase activity calculation formula: enzyme activity (U/mL) ═ nxVt×A/(ε×30×Ve/103) (ii) a n: dilution times of enzyme liquid; a: a change value of absorbance; vt: the total volume of the enzyme reaction solution; ve: volume of enzyme solution in the enzyme reaction solution; epsilon: molar absorptivity.
During the induction period of methanol, the fluorescence value of the bacterial EGFP of the strain GS115-E/X4c during the induction period is gradually increased along with the increase of induction time, the curve is similar to the xylanase enzyme activity curve (figure 5-a), the fluorescence value of the bacterial EGFP and the xylanase enzyme activity value of fermentation supernatant are subjected to correlation analysis, and the correlation coefficient R between the fluorescence value and the xylanase enzyme activity value of the fermentation supernatant is found20.9728 (FIG. 5-b), which shows that the relationship between them is highly correlated (0.8 < | R | < 1), and also shows that the fluorescence quantity of Sec63-EGFP can also be used to characterize the secretion expression quantity of xylanase, the secretion expression quantity of xylanase requires about 60min, and the fluorescence quantity of EGFP for detecting thallus only needs about 60minAt 6min, the secreted expression level of the recombinant xylanase can be rapidly detected by using a fluorescence assay based on Sec 63. Combining the results of examples 2, 3, 4 and 5, it is demonstrated that the fluorescence amount of Sec63-EGFP can be used to characterize the secretory expression amount of recombinant protein, and the secretory expression amount of recombinant protein can be rapidly detected by using Sec 63-based fluorescence analysis method.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
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agatctataa cttcgtataa tgtatgctat acgaacggta ttacttgtac agctcgtcca 60
<210>7
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P7
<400>7
taccgttcgt atagcataca ttatacgaag ttatagatct aacatccaaa gacg 54
<210>8
<211>54
<212>DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<223>P8
<400>8
taccgttcgt ataatgtatg ctatacgaag ttatgtctcc agcttgcaaa ttaa 54

Claims (8)

1. An application of a pichia pastoris strain containing endoplasmic reticulum membrane protein fusion expression fluorescent protein in detecting the secretion expression quantity of pichia pastoris recombinant protein is characterized in that: the endoplasmic reticulum membrane protein is Sec 63.
2. Use according to claim 1, characterized in that:
the detection of the secretion expression quantity of the recombinant pichia pastoris protein is realized by detecting the fluorescence value of the thallus.
3. Use according to claim 1 or 2, characterized in that:
the fluorescent protein is enhanced green fluorescent protein EGFP.
4. Use according to claim 1 or 2, characterized in that:
the pichia is pichia (II)Pichia pastoris）GS115。
5. A fluorescence analysis method for secretion expression quantity of recombinant protein of pichia pastoris is characterized by comprising the following steps: the pichia pastoris endoplasmic reticulum protein is fused to express the fluorescent protein, and the secretion expression quantity of the recombinant protein is represented by detecting the fluorescence value of the thallus; the endoplasmic reticulum membrane protein is Sec 63;
the method specifically comprises the following steps:
(1) constructing a gene segment of endoplasmic reticulum membrane protein-fluorescent protein, and transforming the gene segment into a competent cell of pichia pastoris;
(2) preparing pichia pastoris competent cells of endoplasmic reticulum membrane protein fusion expression fluorescent protein;
(3) transforming a vector for expressing the recombinant protein into the competent cells prepared in the step (2), and coating the competent cells on corresponding screening plates to obtain positive transformants;
(4) inoculating the positive transformant into a fermentation culture medium for fermentation culture, detecting the fluorescence value of thalli of the transformant, and representing the secretion expression quantity of the recombinant protein.
6. The fluorescence analysis method for secretion expression of pichia pastoris recombinant protein according to claim 5, wherein the fluorescence analysis method comprises the following steps:
the fluorescent protein is enhanced green fluorescent protein EGFP;
the pichia is pichia (II)Pichia pastoris）GS115。
7. The application of the fluorescence analysis method for the secretion expression amount of the recombinant pichia pastoris protein according to claim 5 or 6 in detecting the secretion expression amount of the recombinant pichia pastoris protein.
8. Use according to claim 7, characterized in that:
the detection of the secretion expression quantity of the recombinant pichia pastoris protein is realized by detecting the fluorescence value of the thallus.
CN201810922217.6A 2018-08-14 2018-08-14 Fluorescence analysis method for secretion expression quantity of pichia pastoris recombinant protein Active CN109060744B (en)

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