CN106834336B

CN106834336B - Heterologous expression and purification method of trichoderma harzianum acid protease P6281

Info

Publication number: CN106834336B
Application number: CN201710088628.5A
Authority: CN
Inventors: 罗晓春; 邓俊劲; 黄伟谦; 李志伟
Original assignee: South China University of Technology SCUT
Current assignee: South China University of Technology SCUT
Priority date: 2017-02-20
Filing date: 2017-02-20
Publication date: 2020-12-22
Anticipated expiration: 2037-02-20
Also published as: CN106834336A

Abstract

The invention discloses a heterologous expression and purification method of trichoderma harzianum acid protease P6281. Extracting RNA of trichoderma harzianum, performing reverse transcription to obtain cDNA, obtaining an acid protease gene through PCR, inserting the acid protease gene into an expression vector, performing electric transformation on a recombinant plasmid, introducing the recombinant plasmid into pichia pastoris GS115 for induced expression, and performing nickel column affinity chromatography and molecular sieve chromatography to obtain a relatively pure enzyme product. The invention successfully expresses and purifies the Trichoderma harzianum acid protease P6281 for the first time, the method improves the yield of the P6281 protease, and the enzyme activity of the obtained acid protease is far higher than that of the existing Trichoderma acid protease.

Description

Heterologous expression and purification method of trichoderma harzianum acid protease P6281

Technical Field

The invention belongs to the technical field of bioengineering, and particularly relates to a heterologous expression and purification method of trichoderma harzianum acidic protease P6281.

Background

Trichoderma harzianum is a Trichoderma fungus present in almost all soils that readily colonizes plant roots, with some strains having rhizosphere activity, i.e., capable of growing on roots as they develop. Meanwhile, trichoderma harzianum can also obtain nutrition through attacking, parasitizing other fungi and the like. Over the course of many years of evolution, they have developed many mechanisms for attack by other fungi and for enhancing plant and root growth. As an economic, effective, safe and sustainable microbial inoculum, Trichoderma harzianum is also used as a fungicide, is the most important biocontrol bacterium widely applied to biological control of plant mycosis at present, and the bacteriostatic action mechanism and application research thereof have attracted attention. It is used for foliar application, seed treatment and soil treatment to inhibit the growth of various disease causing fungal pathogens. Commercial biotechnological products such as 3Tac, T-22, G-41, etc. currently on the market have been used for the treatment of fungal soil-borne diseases caused by Botrytis, Fusarium and Penicillium. At present, researches speculate that the bacteriostatic mechanism of trichoderma harzianum has competition, heavy parasitism, antibiotics, host immunity stimulation and the like, wherein the heavy parasitism has the most research significance. At present, the research on the mechanism of the parasitism is not clear, and most scientists speculate that the secreted chitinase, glucanase, protease and the like can damage the cell wall of pathogenic fungi, so that the pathogenic fungi can be rooted on the fungi to be parasitized.

In the mechanism research of the trichoderma harzianum parasitism, the research of subarez and the like in 2005 discovers that after cell wall components extracted from botrytis cinerea are added into a culture medium, the expression of various proteins is obviously up-regulated in trichoderma harzianum secretory protein 2D electrophoresis, and the protease P6281 is one of the proteins. Then they obtain the partial sequence of the protease by mass spectrum, then obtain the full length of the gene by PCR, and then obtain the information of theoretical molecular weight, isoelectric point, glycosylation site or active center, etc. of P6281 according to the protein sequence analysis. 61 proteases were found in the sequence of the Surarez pair Trichoderma harzianum EST in 2007, including P6281. In 2009 Samolski also found by microarray chips that trichoderma harzianum also had a large upregulated gene expression in chitin supplemented media, among which was also p 6281. Szabo in 2013 also reported the expression of this gene in trichoderma harzianum parasitized on nematode eggs. It can be seen that P6281 may be an important protease in the process of Trichoderma harzianum parasitizing other fungi and nematode eggs. However, the P6281 protease isolated and purified from Trichoderma harzianum has not been isolated so far, and its properties have not been clearly studied.

However, in the existing reports, the enzyme activity of trichoderma acid protease obtained by heterologous expression and purification is generally not high, for example, the enzyme activity of trichoderma harzianum SA76 acid protease fermentation liquor expressed by Liu in saccharomyces cerevisiae in 2007 is 10.5U/mL, the enzyme activity of neurospora crassa acid protease fermentation liquor expressed by Guo in saccharomyces cerevisiae in 2010 is 6.8U/mL, the enzyme activity of trichoderma asperellum acid protease fermentation liquor expressed by Yang in pichia in 2013 is 18.5U/mL, and the enzyme activity of trichoderma asperellum 55 acid protease pure enzyme liquor expressed by ASP in 2014 Dou is 9.52U/mL.

Disclosure of Invention

The invention aims to overcome the defects of the prior art and provide a heterologous expression and purification method of trichoderma harzianum acid protease P6281. The invention takes trichoderma harzianum as a source, adopts a technical method of genetic engineering to obtain an acid protease gene P6281 from the trichoderma harzianum, constructs a recombinant plasmid, then recombines and expresses in pichia pastoris, purifies an acid protease component in fermentation liquor through affinity chromatography and molecular sieve chromatography, and successfully expresses and purifies the acid protease P6281 of the trichoderma harzianum for the first time.

Another object of the present invention is to provide an acidic protease produced by the method.

The purpose of the invention is realized by the following technical scheme:

a heterologous expression and purification method of Trichoderma harzianum acid protease P6281 comprises the following steps:

(1) culturing trichoderma harzianum;

(2) extracting trichoderma harzianum total RNA;

(3) cloning p6281 coding sequence by RT-PCR;

(4) TA cloning and recombinant plasmid screening and identification;

(5) gene p6281 transforms Pichia pastoris GS 115;

(6) fermentation induction and purification of P6281;

(7) SDS-PAGE detection of recombinant proteins.

The Trichoderma harzianum cultured in the step (1) is to inoculate Trichoderma harzianum GIM3.442 into PDA solid culture medium, and cultured for 2 days at 30 ℃.

The RT-PCR cloning of the p6281 coding sequence in the step (3) comprises the following specific steps: firstly, obtaining Trichoderma harzianum cDNA, then carrying out PCR reaction to obtain a target DNA fragment, wherein the used primers are as follows: forward primer F (5' -CTGC)GAATTCTCGCCGGTAAAGCCAAGT-3 ') and reverse primer R (5' -ACTT)ACGCGTAGCGGCGGTAGCAAAGC-3'), the underlined sequences indicate the EcoR I cleavage site and the MluI cleavage site, respectively.

The specific steps of screening and identifying the TA clone and the recombinant plasmid in the step (4) are preferably as follows: after the high-fidelity enzyme product is recovered by cutting the gel, the gene fragment is connected with a pMD18-T vector, and the T vector connection system is as follows: pMD18-T is 0.5 μ L, p6281 is 4.5 μ L, Solution I is 5 μ L.

The specific steps for transforming the pichia pastoris GS115 by the gene p6281 in the step (5) are preferably as follows: extracting an expression plasmid vector, then carrying out enzyme digestion on the expression plasmid vector by BglII endonuclease, and linearizing the expression plasmid vector; preparing pichia pastoris GS115 competence; the linearized plasmid was transferred into pichia pastoris using an electrical transformation method.

The specific steps for transforming the pichia pastoris GS115 by the gene p6281 in the step (5) are further preferably as follows:

extracting pMD18-T-p6281 cloning vector plasmid, performing PCR amplification by using the pMD18-T-p6281 cloning vector plasmid as a template, and recovering a product after identifying the product to obtain a recovered product;

secondly, transforming the multi-enzyme cutting sites of the expression vector pPIC9K into BamHI, EcoRI, ApaI and MluI in sequence, and constructing to obtain an expression vector pPIC9 BM;

thirdly, after carrying out double enzyme digestion on the recovered product obtained in the step I and the pPIC9BM vector obtained in the step II by using EcoRI and MluI, respectively purifying and recovering to obtain a protease gene fragment subjected to double enzyme digestion and a pPIC9BM vector;

fourthly, the protease gene fragment obtained in the third step is connected with the pPIC9BM carrier in vitro to construct expression plasmid pPIC9BM-p 6281;

fifthly, digesting the expression plasmid pPIC9BM-p6281 by BglII endonuclease, and linearizing the expression plasmid vector;

preparing pichia pastoris GS115 competence; transferring the linearized plasmid obtained in the fifth step into pichia pastoris by using an electrical conversion method.

The identification in step (i) is preferably performed by agarose gel electrophoresis.

The protease gene fragment and the pPIC9BM vector described in the step (iv) are preferably expressed in a molar ratio of 1: 5 proportion for in vitro connection.

The purification described in step (6) is preferably a purification of the protein of interest using a nickel column affinity chromatography and a Sephadex G-75 column chromatography.

The eluent used for the nickel column affinity chromatography is preferably 0.01M imidazole, 0.5M NaCl and 0.02M phosphate buffer (pH7.4); the Sephadex G-75 column chromatography washing solution is preferably 0.05M NaCl and 0.02M phosphate buffer solution (pH6.0).

An acid protease is prepared by the above method.

Compared with the prior art, the invention has the following advantages and effects:

1. the invention successfully expresses and purifies the trichoderma harzianum acidic protease P6281 for the first time.

2. The enzyme activity of the P6281 protease obtained by the invention is far higher than that of the existing acid protease.

The protease activity of P6281 is determined by a national standard method, the protease activity of the fermentation liquor is 321.8U/mL, the specific enzyme activity is 4373.1U/mg, which is higher than the acid protease enzyme activity reported by the same type, for example, the enzyme activity of trichoderma harzianum SA76 acid protease fermentation liquor expressed by Liu in saccharomyces cerevisiae in 2007 is 10.5U/mL, the enzyme activity of neurospora crassa acid protease fermentation liquor expressed by Guo in saccharomyces cerevisiae in 2010 is 6.8U/mL, the enzyme activity of trichoderma asperellum acid protease fermentation liquor expressed by Yang in pichia in 2013 is 18.5U/mL, and the enzyme activity of trichoderma asperella ASP55 acid protease pure enzyme liquor expressed by ASP Dou in escherichia coli in 2014 is 9.52U/mL.

3. The method greatly improves the yield of the P6281 protease.

Drawings

FIG. 1 is a plasmid map of the recombinant expression vector pPIC9BM-p 6281.

FIG. 2 is a SDS-PAGE gel electrophoresis of recombinant proteins, wherein M represents a standard molecular weight protein, lane 1 is a crude enzyme solution obtained after 4 days of fermentation induction, lane 2 is a sample purified after 5 days of fermentation induction, lane 3 is a crude enzyme solution obtained after 5 days of fermentation induction, and lane 4 is a Pichia pastoris supernatant transformed with an empty vector after 5 days of induction.

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.

Examples

Firstly, culturing trichoderma harzianum:

trichoderma harzianum GIM3.442 (purchased from Guangdong province culture Collection) was inoculated into a PDA solid medium (200 mL of potato powder filtrate, 4g of glucose, 0.75g of magnesium sulfate, 0.75g of potassium dihydrogen phosphate, and 4g of agar powder) and cultured at 30 ℃ for 2 days.

(II) extracting the total RNA of trichoderma harzianum:

(1) taking 100mg of mycelia with forceps sterilized by high pressure steam, placing into a mortar precooled by liquid nitrogen, adding a small amount of liquid nitrogen, rapidly grinding with the mortar, adding a small amount of liquid nitrogen, continuously grinding, and repeating for 3 times until all mycelia completely become white powder.

(2) 2mL of RNAioso Plus (available from Dalibao bioengineering, Inc.) was added to the mortar to cover the powder as completely as possible, and then allowed to stand at room temperature until the RNAioso Plus was completely melted, and the grinding was continued with the mortar until the lysate became transparent. The resulting lysate was transferred in equal amounts to a 1.5mL centrifuge tube and allowed to stand at room temperature for 5 minutes. Centrifuge at 12000rpm for 5 minutes at 4 ℃ and carefully aspirate the supernatant and transfer it to a new centrifuge tube (do not aspirate the pellet).

(3) To the supernatant obtained in step (2), 400. mu.L of chloroform was added, and the centrifuge tube cap was closed and vigorously shaken for 15 seconds. After the solution was emulsified sufficiently, it was allowed to stand at room temperature for several minutes, and then centrifuged at 12000rpm at 4 ℃ for 15 minutes.

(4) The centrifuge tube was carefully removed from the centrifuge and the homogenate was now divided into three layers, a colorless supernatant, a middle white protein layer and a colored lower organic phase. Sucking the supernatant and transferring the supernatant into another new centrifuge tube;

(5) adding isopropanol with the same volume into the supernatant obtained in the step (4), turning the centrifuge tube upside down, fully mixing the mixture, standing the mixture at room temperature for 10 minutes, and then centrifuging the mixture at 12000rpm at 4 ℃ for 10 minutes.

(6) After centrifugation, the bottom of the tube had a pellet. The supernatant was carefully discarded, 1mL of 75% ethanol (without touching the pellet) was slowly added along the walls of the tube, the tube walls were gently inverted, washed at 12000rpm, centrifuged at 4 ℃ for 5 minutes and the ethanol carefully discarded.

(7) And opening the cover of the centrifugal tube, inverting the centrifugal tube, drying the precipitate at room temperature for 5 minutes, adding 20 mu L of RNase-free water to dissolve the precipitate, transferring the dissolved solution into the RNase-free centrifugal tube after the precipitate is completely dissolved, and storing at-80 ℃.

(III) RT-PCR cloning of p6281 coding sequence:

(1) the following components were added to a ribozyme-free PCR tube:

TABLE 1RT-PCR System

(2) Incubating the mixture at 65 ℃ for 5 minutes, rapidly freezing the mixture on ice, adding 4. mu.L of 5 XFirst-Strand buffer, 2. mu.L of 0.1M DTT, 1. mu.L of 40U/mL RNase inhibitor to the mixture, gently mixing and incubating the mixture at 37 ℃ for 2 minutes;

(3) adding 1 μ L of reverse transcriptase, mixing gently, and culturing at 37 deg.C for 50 min;

(4) culturing at 70 deg.C for 15 min to inactivate reverse transcriptase, and storing at-20 deg.C;

(5) a forward primer F (5'-CTGCGAATTCTCGCCGGTAAAGCCAAGT-3') and a reverse primer R (5'-ACTTACGCGTAGCGGCGGTAGCAAAGC-3') were designed. Performing PCR reaction by using the Trichoderma harzianum cDNA obtained in the previous step as a template according to the following PCR system and program to obtain a target DNA fragment;

TABLE 2PCR System

The PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds; annealing at 51 ℃ for 30 seconds; extension at 72 ℃ for 2 min; 32 cycles; final extension at 72 ℃ for 10 min; the product was then identified by agarose gel electrophoresis at an agarose concentration of 1.5% under conditions of 120V for 25min, the procedure for agarose gel electrophoresis being described in molecular cloning, a laboratory Manual. The size of the obtained acid protease gene band is about 1100bp, and a gel recovery kit is used for recovering the target gene.

(IV) TA cloning and recombinant plasmid screening identification

(1) After the high-fidelity enzyme product is cut and recovered, Ex-taq enzyme is utilized to carry out A addition reaction, and the target gene fragment (p6281) obtained in the step (III) is connected with a pMD18-T vector (purchased from Dalibao bioengineering Co., Ltd.) according to the following system under the connection condition of 16 ℃ for 2 hours;

TABLE 3T Carrier ligation systems

Then, E.coli DH 5. alpha. competent cells (purchased from Dalibao bioengineering Co., Ltd.) were transformed by heat shock at 42 ℃ for 70 seconds, spread on LB liquid medium (tryptone 10g, yeast extract 5g, sodium chloride 10g, distilled water to a volume of 1000mL) containing ampicillin resistance, and cultured overnight at 37 ℃. Then colony PCR is carried out by using 2 xTaq PCR Mix to screen positive colonies, and the reaction system and the program are as follows:

TABLE 4 colony PCR System

The colony PCR reaction conditions are as follows: pre-denaturation at 94 ℃ for 3 min; denaturation at 94 ℃ for 30 seconds; annealing at 51 ℃ for 30 seconds; extension at 72 ℃ for 1 min; 32 cycles; final extension at 72 ℃ for 10 min; then, identifying the product by agarose gel electrophoresis, wherein the agarose concentration is 1%, the electrophoresis condition is 120V, and the size of the obtained acid protease gene band is about 1100bp in 25 min;

(2) the positive clones were picked and added to LB liquid medium containing ampicillin resistance for 12h on a shaker at 37 ℃ and 220 rpm. Taking the enlarged culture bacterial liquid in a centrifuge tube, sending the liquid to biological engineering (Shanghai) corporation for sequencing, and determining the length of the cloned gene to be 1107bp through sequencing, wherein the sequencing result is as follows: the nucleotide sequence is shown as SEQ ID No. 3;

(V) gene p6281 transforming Pichia pastoris GS 115:

(1) extracting pMD18-T-p6281 clone carrier plasmid with cloned acid protease gene, PCR amplifying with the plasmid as template, referring to the above table 2, then agarose gel electrophoresis identifying the product, purifying and recovering with kit. The expression vector pPIC9K (purchased from Invitrogen) was modified in multiple enzymatic cleavage sites, in the order BamHI, EcoRIApaI and MluI, and designated pPIC9BM (FIG. 1). The vector was constructed using RF cloning with primers 5' -GAAGCTGGATCCGAATTCCCGCTCGAGGGGCCCACGCGTCATCATCATCATC-3' (the underlined sequences represent the EcoRI and MluI cleavage sites, respectively), the reaction system is:

TABLE 5RF cloning System

The reaction conditions are as follows: pre-denaturation at 98 ℃ for 3 min; denaturation at 98 ℃ for 10 seconds; annealing at 58 ℃ for 15 seconds; extension at 72 ℃ for 8 min; 32 cycles; final extension at 72 ℃ for 10 min; sequencing and identifying a product;

carrying out double enzyme digestion on the recovered product and the pPIC9BM vector by using EcoRI and MluI according to the instruction (the double enzyme digestion system is shown in Table 6), respectively purifying and recovering the DNA after enzyme digestion by using a common DNA recovery kit, and then detecting the DNA concentration by using a nucleic acid concentration detector;

TABLE 6 double enzyme digestion System

The enzyme digestion condition is 37 ℃ and 4 hours;

(2) and (3) purifying and recovering the protease gene fragment p6281 and the pPIC9BM vector fragments according to the molar ratio of 1: 5 in vitro ligation was carried out using T4 ligase at 22 ℃ for 8h, in the following table.

TABLE 7T4 ligase ligation

The ligated recombinant plasmid was transformed into E.coli DH 5. alpha. strain by heat shock at 42 ℃ for 70 seconds. Selecting positive single colony on the plate, extracting plasmid to perform double enzyme digestion identification, and performing bacterial liquid and plasmid sequencing identification on the strain; the successfully constructed expression plasmid was designated pPIC9BM-p 6281.

(3) Extracting an expression plasmid vector from the bacterial suspension of the positive clone bacterial strain by adopting a plasmid miniextraction kit, then carrying out enzyme digestion on the expression plasmid vector by BglII endonuclease, and linearizing the expression plasmid vector, wherein the enzyme digestion system is as follows:

TABLE 8 Single enzyme digestion System

After enzyme digestion, the plasmid is recovered by a recovery kit and the concentration is measured;

(4) the preparation method of pichia pastoris GS115 competence comprises the following specific steps:

a. inoculating a Pichia pastoris GS115 strain frozen at-80 ℃ on a YPD plate (tryptone 20g, yeast extract 10g, glucose 20g, agar powder 20g, distilled water constant volume to 1000mL) by streaking, and culturing at 30 ℃ for 3 days;

b. selecting a single colony of the Pichia pastoris GS115 strain, inoculating the single colony into a 250mL triangular flask containing 25mL LYPD culture solution (tryptone 20g, yeast extract 10g, glucose 20g and distilled water with constant volume of 1000mL), and carrying out shaking culture at 30 ℃ and 220rpm for 2 days;

c. b, inoculating 1mL of the bacterial suspension finally obtained in the step b into another flask containing 50mLYPD culture solution, and carrying out shaking culture at 30 ℃ and 220rpm for several hours until the OD600 of the bacterial suspension reaches 2.0;

d. transferring the bacterial suspension into a sterilized 50mL centrifuge tube, centrifuging at 5000rpm and 4 ℃ for 5 minutes, removing supernatant, re-suspending the thallus by 10mL of precooled sterile water, and transferring into a 15mL centrifuge tube;

e.5000rpm, centrifuging for 5 minutes at 4 ℃, removing supernatant, re-suspending the thalli by 10mL of precooled 1M sorbitol, and repeating the steps once;

f.5000rpm, centrifugation at 4 ℃ for 5 minutes, supernatant removal, 1mL of 1M sorbitol heavy suspension of the thalli, and placing on ice for electric transformation;

(5) transferring the linearized plasmid obtained in the step (3) into pichia pastoris by using an electrical transformation method, wherein the electrical shock condition is 1.5kV, 2mm electrical rotating cups and 10ng of linearized plasmid. Coating the electrically transformed bacterial liquid on an MD plate, culturing for 2 days at 30 ℃, selecting 5 single colonies, respectively inoculating to a YPD liquid culture medium, then cracking pichia pastoris transformant cells at low temperature,

the cracking method comprises the following steps:

a. selecting 10 pichia pastoris transformant single colonies from an MD plate (glucose 20g, YNB 13.4g, agar powder 20g and distilled water with constant volume of 1000mL), respectively inoculating into 2mLYPD culture solution, and carrying out shake culture at 30 ℃ and 220rpm for 2 days;

b. respectively transferring 1mL of bacterial liquid into a centrifuge tube, centrifuging at 8000rpm for 2min, and discarding the supernatant;

c. adding 1mL of TE buffer to resuspend the thalli, centrifuging at 8000rpm for 2min, discarding the supernatant, and repeating once;

d. transferring to a refrigerator at-80 ℃ for one hour after 30min of boiling water bath, and then carrying out 10min of boiling water bath;

e.8000rpm for 2min, and storing the obtained supernatant at-20 ℃;

2 XTaq PCR Mix was used for colony PCR identification, the reaction system and program are referred to Table 4;

(VI) fermentation induction and purification of P6281:

the positive recombinant pichia pastoris strain is selected, streaked on an MD plate, cultured at 30 ℃ for 2 days, a single colony is selected and inoculated into a triangular flask containing 50mL of BMGY culture solution (yeast extract 10g, tryptone 20g, YNB 13.4g, glycerol 10mL, 1M potassium phosphate (pH6.0)100mL, distilled water constant volume is 1000mL), and the culture is carried out at 30 ℃ and 220rpm in a shaking way until OD600 is approximately equal to 5.0. Then, the thalli is collected by centrifugation, thalli precipitates are transferred into a triangular flask filled with 100mL of BMMY culture solution in an equivalent manner, the culture is carried out at 28 ℃ and 220rpm in a shaking way, 1.5% methanol solution is added every 24 hours for induction expression, and the induction lasts for 4-5 days. Centrifuging the fermentation liquor at 5000rpm and 4 deg.C for 10min to obtain supernatant for measuring enzyme activity; the protease activity of P6281 was determined by the "Folin method" in SB/T10317-1999 protease Activity assay. Then purifying the target protein by using nickel column affinity chromatography and Sephadex G-75 column chromatography, wherein the sample amount of the nickel column affinity chromatography is 150mL, and the eluent is 0.01M imidazole, 0.5M NaCl and 0.02M phosphate buffer (pH 7.4); the loading amount of Sephadex G-75 column chromatography was 95mL, and the washing solution was 0.05M NaCl and 0.02M phosphate buffer (pH 6.0). The GS115 strain transformed with the empty vector pPIC9BM was used as an experimental control.

By the method, the protease activity of the fermentation liquid obtained by the invention is 321.8U/mL, and the specific enzyme activity is 4373.1U/mg. The yield of the purified protease was 116.5mg/1000mL of the fermentation broth as determined by BCA protein concentration kit of Biotechnology engineering (Shanghai) Ltd.

Protein expression was studied in the case of Trichoderma harzianum, which expresses P6281 in the largest amount but expresses only 18. mu.g/300 mL of total protein (including P6281, unpurified) under conditions in which the B.cinerea cell wall was added, by adding 1% of P.ultimum, B.cinerea, R.solani cell wall and chitin as carbon sources, respectively. Therefore, the invention not only successfully expresses and purifies the heterologous gene for the first time to obtain the P6281, but also obviously improves the yield.

(seventhly) SDS-PAGE detection of recombinant proteins:

SDS-PAGE gel electrophoresis is used to confirm the expression, purity and molecular mass of the recombinant protease. The concentration of the adopted concentrated gel is 12 percent and the concentration of the adopted separation gel is 5 percent, the loading amount is 20 mu L, and standard protein with standard molecular weight is used as Marker. The operation of SDS-PAGE gel electrophoresis is described in protein electrophoresis experiment. For the preparation of fermentation liquor samples, the amount of recombinant protease generated by induction expression is high, the fermentation liquor can be directly diluted by 1 time and then is uniformly mixed with a loading buffer solution, after boiling for 10min with boiling water, the mixture is centrifuged at 12000rpm for 1min, and electrophoresis is carried out after loading.

SDS-PAGE electrophorograms of the crude enzyme solution (which refers to fermentation liquor which is not subjected to nickel column affinity chromatography and Sephadex G-75 column chromatography) and the purified enzyme solution are shown in FIG. 2, wherein M represents a standard molecular weight protein, lane 1 is the crude enzyme solution obtained after 4 days of fermentation induction, lane 2 is a sample purified by affinity chromatography and molecular sieve chromatography after 5 days of fermentation induction, lane 3 is the crude enzyme solution induced by 5 days of fermentation, and lane 4 is the Pichia pastoris supernatant of the converted empty vector after 5 days of induction. As can be seen, the empty vector transformed Pichia pastoris had no protein expression, while the unpurified positive induction (i.e., lanes 1 and 3) supernatants contained two protein bands, one dark and one light, with the dark band size approaching 40kDa, consistent with the expected results; while the protein expression level induced for 5 days was greater than 4 days, lane 2 shows that only a single expected protein band remained after purification, indicating that the electrophoretically pure level of acid protease P6281 was successfully obtained.

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.

SEQUENCE LISTING

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tcggtcacta ttggtggtaa atcctgggac ctcatcgtcg acactggatc ttcaaacacg 240

tggtgtggtg ctcaaagctc atgcgagcct tcatctactg gcaagtccac gggcggttcc 300

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aacgccaaca cggttccaac cttcttggat aatctctaca gccaaggttc catctcgact 540

gaggtgctgg gcgtttcttt caagccagag tctggcagtg acagtgatga caccaacggc 600

gagttgaccc tcggcggtac tgatagctcc aagtacacgg gctctctcac ctacttctca 660

actctcaaga gtggctctgc tgctccctac tggggcatct ctattgctag tttcacctac 720

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tacggactca gctctggaaa gtactacgct tggattaacg acggtggcag ctcgggtgtc 1020

aacaccatta ttggccagaa gttcctggaa aactactact ccgtttttga tactaccaac 1080

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Claims

1. A heterologous expression and purification method of Trichoderma harzianum acid protease P6281 is characterized by comprising the following steps:

(1) culturing trichoderma harzianum;

(2) extracting trichoderma harzianum total RNA;

(3) cloning p6281 coding sequence by RT-PCR;

(4) TA cloning and recombinant plasmid screening and identification;

(5) the screened and identified recombinant plasmid is transformed into pichia pastoris GS 115;

(6) fermentation induction and purification of P6281 recombinant protein, wherein the specific operation of the fermentation induction is as follows: transferring the positive recombinant pichia pastoris to BMGY culture solution, and adding 1.5% methanol solution for induction expression;

(7) SDS-PAGE detection of P6281 recombinant protein;

the RT-PCR cloning of the p6281 coding sequence described in step (3) was performed as follows: firstly, obtaining Trichoderma harzianum cDNA, then carrying out PCR reaction to obtain a target DNA fragment, wherein the used primers are as follows: a forward primer F: 5'-CTGCGAATTCTCGCCGGTAAAGCCAAGT-3' and reverse primer R: 5'-ACTTACGCGTAGCGGCGGTAGCAAAGC-3', respectively;

the step of transforming the recombinant plasmid screened and identified in the step (5) into pichia pastoris GS115 is as follows: extracting recombinant plasmids, then carrying out enzyme digestion on the recombinant plasmids by BglII endonuclease, and linearizing the recombinant plasmids; preparing pichia pastoris GS115 competence; transferring the linearized plasmid into pichia pastoris by using an electrical transformation method;

the trichoderma harzianum is GIM 3.442;

the steps for screening and identifying the TA clone and the recombinant plasmid in the step (4) are as follows: after the high-fidelity enzyme product is recovered by cutting the gel, the gene fragment is connected with a pMD18-T vector, and the T vector connection system is as follows: pMD18-T is 0.5 μ L, p6281 is 4.5 μ L, Solution I is 5 μ L.

2. The method for heterologous expression and purification of trichoderma harzianum acidic protease P6281 of claim 1, wherein said culturing trichoderma harzianum in step (1) is performed by inoculating trichoderma harzianum GIM3.442 into PDA solid medium and culturing at 30 ℃ for 2 days.

3. The method for heterologous expression and purification of trichoderma harzianum acid protease P6281 according to claim 1, wherein the purification in step (6) is a purification of the target protein using nickel column affinity chromatography and Sephadex G-75 column chromatography.