CN104694560A - Disulfide bond isomerase gene Trpdi2 from Trichoderma reesei and application thereof - Google Patents

Abstract

The invention discloses a disulfide bond isomerase gene Trpdi2 derived from Trichoderma reesei and its application. The disulfide bond isomerase gene isolated from Trichoderma reesei in the present invention has a The nucleotide sequence is shown in (a) or (b): (a) the polynucleotide sequence shown in SEQ ID No.1; (b) is complementary to the polynucleotide sequence in (a) according to the principle of base complementary pairing Polynucleotide sequence and (c) the sequence of the polynucleotide sequence shown in SEQ ID NO.1 (a) or the cDNA sequence of the polynucleotide sequence in (b): the polynucleotide sequence shown in SEQ ID NO.2 . The invention also obtains the disulfide bond isomerase from the coded disulfide bond isomerase gene isolated from Trichoderma reesei and verifies the application of the disulfide bond isomerase.

Description

A disulfide bond isomerase gene Trpdi2 derived from Trichoderma reesei and its use

technical field

The invention relates to the fields of biochemical engineering and microbial genetic engineering, in particular to a disulfide bond isomerase gene Trpdi2 derived from Trichoderma reesei and its application.

Background technique

Due to the cellulase resources and their cellulase production capacity, filamentous fungi have received great attention in the past few decades, but with the development of industry and biomedicine, people hope to use filamentous fungi with strong secretion ability Fungi act as cell factories to more efficiently produce endogenous and exogenous proteins.

Compared with other expression receptors, filamentous fungi have greater advantages as expression receptors of foreign proteins. Compared with prokaryotic expression hosts, filamentous fungi can perform post-translational processing of foreign proteins such as disulfide bond formation, glycosylation, and protease cleavage; compared with yeast, the glycosylation of filamentous fungi is closer to that of mammals. It can avoid excessive glycosylation of foreign proteins by yeast; compared with mammalian cells, insects and other higher biological expression systems, filamentous fungi have the advantages of fast growth, lower cost, and easier scale formation (Zhong Yaohua et al., 2010) . In addition, industrial filamentous fungi such as Trichoderma reesei, Aspergillus niger, and Aspergillus oryzae have been identified as GRAS (Generally Recognized as Safe) and food additive strains by the US Food and Drug Administration, ensuring their safety as host strains (Zhang Jianjun et al., 2009; Liu Dehui et al., 2010).

Among them, Trichoderma reesei is widely used in industry, and its own protein secretion can reach 100g/L, but as a host to express foreign protein, the output is only maintained at the milligram level, which is mainly due to transcription, translation, post-translational modification and It is caused by the restriction of multi-level factors such as secretion (Nevalainen et al., 2005). In order to overcome these limitations, people have also carried out some work, mainly focusing on the operation of transcription level such as using strong promoters, increasing gene copy number, and fusion expression with endogenous genes. Although the results show that the product has increased to a certain extent, it is limited. The degree of improvement also indicates that the level of transcription is not the main limiting factor, and the current major research is turning to post-translational processes.

During the process of self and foreign proteins being translated and secreted outside the cell, the quality of the protein will be monitored, and only correctly folded proteins with the correct structure can be successfully secreted (Saloheimo et al., 2012). The formation of disulfide bonds is extremely important in the protein folding process. Disulfide bond is a covalent link between two cysteines in a protein polypeptide chain or between chains. Its formation is an important step in the folding and maturation of oxidized proteins. It can improve protein stability and reduce proteases. It can degrade protein and have important influence on the structure and biological activity of protein. The mechanism of the influence of disulfide bonds on protein activity is mainly divided into three types: 1. The cysteine that forms the disulfide bond is the active site of the enzyme, and its redox state determines the catalytic reaction of the enzyme; 2. The disulfide bond is close to the activity 3. Maintain the specific conformation of the protein through the disulfide bond to ensure the function (Wang Liang et al., 2011). A lot of evidence has confirmed that disulfide bonds can be formed spontaneously in vitro, but the speed is much lower than that of in vivo reactions, because the formation of disulfide bonds in vivo is catalyzed by enzymes, and electrons are passed between cysteine and disulfide bonds. The transmission is realized. The disulfide isomerase system present on the endoplasmic reticulum is responsible for disulfide bond formation in T. reesei and is important for the maturation of T. reesei proteins.

When a large amount of endogenous or exogenous proteins are expressed, the post-translational process of Trichoderma reesei is overloaded, resulting in a relative lack of enzymes in the endoplasmic reticulum, which will cause protein misfolding and assembly. By expressing the disulfide bond isomerase gene, the ability of disulfide bond formation during protein maturation can be improved, and finally the production of exogenous protein can be increased (Sha et al., 2013; Mukaiyama et al., 2010). Therefore, the identification and cloning of highly active disulfide bond isomerase genes can provide tools for increasing the ability of Trichoderma reesei to express foreign proteins and restoring the activity of disulfide bond essential proteins in vitro.

Contents of the invention

One of the objectives of the present invention is to provide a disulfide bond isomerase-based Trpdi2 derived from Trichoderma reesei.

The second object of the present invention is to provide an expression vector and a recombinant host cell containing the above-mentioned disulfide bond isomerase gene Trpdi2 derived from Trichoderma reesei.

The third object of the present invention is to provide the use of the disulfide bond isomerase encoded by the disulfide bond isomerase gene Trpdi2 derived from Trichoderma reesei.

The invention discloses a disulfide bond isomerase gene isolated from Trichoderma reesei, characterized in that its polynucleotide sequence is shown in (a) or (b):

(a) the polynucleotide sequence shown in SEQ ID No.1;

(b) A polynucleotide sequence complementary to the polynucleotide sequence in (a) according to the principle of complementary base pairing.

Preferably, its polynucleotide sequence is also shown in (c):

(c) The polynucleotide sequence shown in SEQ ID NO.1 (a) or the cDNA sequence of the polynucleotide sequence in (b): the polynucleotide sequence shown in SEQ ID NO.2.

A disulfide bond isomerase encoded by the disulfide bond isomerase gene isolated from Trichoderma reesei as claimed in claim 1.

Preferably, the amino acid sequence of the disulfide bond isomerase is the amino acid sequence shown in SEQ ID NO.3.

An expression vector containing the disulfide bond isomerase gene isolated from Trichoderma reesei as claimed in claim 1. The expression vector is a recombinant expression vector for expressing foreign genes in bacteria or fungi and a recombinant host strain or transformant containing the recombinant expression vector; the recombinant expression vector includes: a promoter, a disulfide bond heterogeneous The polynucleotide sequence of the isomerase gene and the terminator; wherein, the polynucleotide sequence of the disulfide bond isomerase gene is located downstream of the promoter, and the terminator is located downstream of the exogenous gene sequence to be transcribed.

A recombinant host cell containing the expression vector of the disulfide bond isomerase gene according to claim 1.

Preferably, the application of the disulfide bond isomerase to increase the expression of endogenous or exogenous proteins.

Preferably, the disulfide bond isomerase is used in stabilizing and restoring disulfide bond essential enzyme activity.

The beneficial effect of the present invention is that the present invention proves that there is a gene Trpdi2 that plays an important role in disulfide bond formation in Trichoderma reesei by using biological methods including in vitro protein expression and protein activity assay, and the polynucleotide of the gene Trpdi2 is clarified sequence, and the polynucleotide sequence of its cDNA, and provides the amino acid sequence of the disulfide bond isomerase derived from the disulfide bond isomerase gene coded by Trichoderma reesei; the disulfide bond isomerase can be found in It catalyzes the disulfide bond formation of proteins from different sources in vitro, and is a new type of disulfide bond isomerase. The disulfide bond isomerase can also be used to restore or maintain the activity of disulfide bond essential enzymes in vitro, and can be used to improve the yield of exogenous protein expressed by Trichoderma reesei.

Definition of terms involved in the present invention

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices and materials are now described.

The term "principle of complementary base pairing" means that in the molecular structure of DNA, since the hydrogen bonds between bases have a fixed number and the distance between the two strands of DNA remains constant, base pairing must follow certain rules , that is, Adenine (A, adenine) must be paired with Thymine (T, thymine), and Guanine (G, guanine) must be paired with Cytosine (C, cytosine), and vice versa.

The term "expression vector" means a vector that adds expression elements (such as promoter, RBS, terminator, etc.) on the basis of the basic skeleton of the cloning vector to enable the expression of the target gene. For example, the expression vector pKK223-3 is an E. coli expression vector with a typical expression structure. Its basic backbone is the plasmid origin of replication and ampicillin resistance gene from pBR322 and pUC. In the expression element, there is a hybrid tac strong promoter and terminator, and there is an RBS site downstream of the promoter (if this site is used, the distance between ATG and ATG is required to be 5-13 bp), followed by the multiple cloning site The gene of interest to be expressed can be loaded. The target gene in the present invention is the cDNA of the polynucleotide sequence (a) or (b) in the polynucleotide sequence shown in SEQ ID NO.1.

The term "recombinant host cell" means a recipient cell of a plasmid vector comprising the strong promoter RPL of the present invention and a terminator corresponding thereto, regardless of the method used for insertion to produce a recipient cell, such as direct suction, transduction or Other methods known in the art.

The term "polynucleotide" means deoxyribonucleotides, deoxyribonucleosides (DNA), ribonucleosides or ribonucleotides (RNA) and polymers thereof in single- or double-stranded form. "DNA" or "cDNA" of the disulfide bond isomerase gene as described in the present invention and the like. Unless specifically limited, the term encompasses nucleic acids that contain known analogs of natural nucleotides that have binding properties similar to the reference nucleic acid and are metabolized in a manner similar to naturally occurring nucleotides. Unless specifically limited otherwise, the term also means oligonucleotide analogs, including PNA (peptide nucleic acid), DNA analogs used in antisense technology (phosphorothioate, phosphoramidate, etc.) . Unless otherwise specified, a particular nucleic acid sequence also implicitly encompasses conservatively modified variants thereof (including, but not limited to, degenerate codon substitutions) and complementary sequences as well as the explicitly designated sequences. In particular, degenerate codon substitutions can be achieved by generating sequences in which one or more selected (or all) codons are substituted at position 3 with mixed bases and/or deoxyinosine residues (Batzer et al. , Nucleic Acid Res.19:5081 (1991); Ohtsuka et al., J.Biol.Chem.260:2605-2608 (1985); and Cassol et al., (1992); Rossolini et al., Mol Cell.Probes8:91 -98(1994)). Wherein, the cDNA of the disulfide bond isomerase gene mentioned in the present invention is reverse-transcribed from the RNA obtained after the DNA of the disulfide bond isomerase gene is transcribed.

The term "amino acid" means the basic unit of protein, which endows the protein with a specific molecular structure and makes its molecules biochemically active. Proteins are important active molecules in organisms, including enzymes and enzymes that catalyze metabolism. Different amino acids are dehydrated and condensed to form peptides (the original fragments of proteins), which are the precursors of protein production.

The terms "endogenous protein", "exogenous protein" and "protein" mean a polymer of amino acid residues. The term applies to naturally occurring amino acid polymers as well as amino acid polymers in which one or more amino acid residues is a non-naturally encoded amino acid.

Description of drawings

Fig. 1 is the electrophoresis diagram of the disulfide bond isomerase gene Trpdi2 and the vector used for the expression of Trpdi2.

Fig. 2 is the electrophoresis pattern of disulfide bond isomerase gene Trpdi2 in Escherichia coli BL21 (DE3) after induced expression and nickel column purification on SDS polypropylene gel.

Figure 3 is the result of disulfide bond isomerase activity assay.

Figure 4 shows the effect of different concentrations of DTT on the activity of Trichoderma reesei exocellulase CBH1.

Figure 5 is a graph showing the results of disulfide bond isomerase recovery of the activity of reductively inactivated Trichoderma reesei exocellulase CBH1.

Detailed ways

The present invention will be further described in detail below in conjunction with the accompanying drawings, so that those skilled in the art can implement it with reference to the description.

Example 1: Cloning of Disulfide Bond Isomerase Gene

(1) Cloning of Trpdi2 Genomic DNA

According to the sequence information of SEQ ID: 1 in the Trichoderma reesei data (http://genome.jgi-psf.org/Trire2/Trire2.home.html), design relevant primers, the primer sequence is:

Trpdi2-F: ATGGTCTTGATCAAGAGCCTC,

Trpdi2-R: TCACAGCTCGTCCTTCTGG,

Using the genomic DNA of the QM9414 strain stored at -20°C as a template, PCR amplification was performed to obtain the full-length DNA fragment of the gene, which was cloned into the pGEM-T vector.

Wherein, the PCR reaction system is:

The PCR reaction conditions are:

(2) Cloning of Trpdi2 cDNA

Using the cDNA of QM9414 strain stored at -80°C as a template, the cDNA fragment was amplified by PCR with primers Trpdi2-F and Trpdi2-R, and cloned into pGEM-T vector.

in,

The PCR reaction system is:

The PCR reaction conditions are:

Example 2: Expression and Activity Measurement of Disulfide Bond Isomerase

(1) Expression vector construction of disulfide bond isomerase gene Trpdi2

According to the sequence of the disulfide bond isomerase gene Trpdi2, primers with restriction sites at the end were designed, and the cDNA of the QM9414 strain stored at -80°C was used as a template to perform PCR amplification to obtain the cDNA containing the sequence of the disulfide bond isomerase gene Trpdi2 DNA fragments, DNA fragments recovered by double digestion with NcoI and BamHI and the pET-24d vector preserved in our laboratory, as shown in Figure 1. The two restriction fragments were recovered and ligated to obtain an expression vector, and the obtained expression vector was transformed into Escherichia coli DH5α. The transformed Escherichia coli DH5α was sequenced and detected, and the sequenced detection proved that the transformed DH5α was correct, and the extracted plasmid was transformed into Escherichia coli BL21(DE3). In Figure 1, Trpdi2 represents the disulfide bond isomerase gene after double digestion, and pET-24d is the expression vector of double digestion.

Among them, the connection conditions for constructing the expression vector are:

(2) Expression of disulfide bond isomerase TrPDI2

Pick a BL21-positive single colony containing the pET24d-Trpdi2 plasmid, inoculate it into 5 mL of LB medium containing kanamycin, and culture it with shaking at 37°C overnight; take 1 mL of the bacterial liquid and inoculate it into 100 mL of LB medium containing kanamycin , shake culture at 37°C until OD600=0.6-0.8; add IPTG to a final concentration of 0.2mM, shake culture at 16°C, 150rpm for 8h.

(3) Purification of disulfide bond isomerase TrPDI2

The LB medium of BL21-positive Escherichia coli containing the pET24d-Trpdi2 plasmid that was cultured for 8 hours by shaking above was collected by centrifugation to collect BL21 cells, and the BL21 cells were crushed by a high-pressure crusher, and the supernatant and precipitate were separated after centrifugation, and the supernatant and The precipitations were subjected to polyacrylamide gel electrophoresis SDS-PAGE, and the position of detecting the disulfide bond isomerase of the target protein was relatively concentrated in the supernatant. Afterwards, the supernatant was used as a sample to purify the target protein through a nickel column, and then the target protein was eluted with gradient eluents of different concentrations of imidazole, and the pure protein was obtained and then concentrated and collected to obtain the target protein. As shown in Figure 2, it is the electrophoresis diagram of the target protein in the sample, the flow-through solution and the eluent of different concentrations of imidazole, wherein the sample is the supernatant, and the flow-through solution is the column solution after the supernatant flows through the nickel column , and concentrations of 20mM, 50mM, 100mM, 150mM, and 200mM imidazole eluents respectively. It can be seen from Figure 2 that the molecular weight of the target protein is 40kDa. High, and different concentrations of imidazole eluate only contain the target protein, to achieve the purpose of purifying the target protein.

(4) Determination of disulfide bond isomerase TrPDI2 activity

Dissolve 5mg of RNaseA in 1mL of 6M guanidine hydrochloride and 0.14M DTT solution, place it overnight at room temperature to reduce and inactivate RNaseA, and the treated RNaseA protein is purified through a desalting column. In the case of glycide and substrate cCMP, the change of A296 was measured to reflect the degree of renaturation of RNaseA, and the reactivity of PDI2 to RNaseA was measured. As shown in Figure 3, the left side is the SDS polyacrylamide gel electrophoresis pattern of the TrPDI2 protein sample used in the activity assay experiment, and the right side is the disulfide bond isomerase activity detection result of TrPDI2, wherein the curve in Figure 3 represents the The progress of the reaction, the change of the absorbance of the reaction solution at 296nm, reflects the degree of the substrate cCMP being acted on by the active ribonuclease A to generate the product. It can be seen from Figure 3 that RNaseA that has not been reductively inactivated by adding TrPDI2 has basically no activity, while adding TrPDI2 can restore part of the activity of RNaseA, indicating that TrPDI2 has disulfide bond isomerase activity.

Example 3: Effect of Disulfide Bond Isomerase on Exoglucanase Activity

(1) Effect of disulfide bond on the activity of exoglucanase CBH1 protein

Using Trichoderma reesei fermentation liquid as a sample, CBH1 pure protein was separated and purified by DEAE protein chromatography column. CBH1 was treated with different concentrations of DTT, and the activity of CBH1 protein was measured after 1, 2, 3, 4, 5, 6 and 18 hours of treatment. The activity was determined based on the activity of the substrate pNPC. As shown in Figure 4, the left side is the SDS polyacrylamide gel electrophoresis image of the purified CBH1 protein sample, and the right side is the result of CBH1 activity after DTT treatment for different times, and the three curves represent the pNPC of CBH1 after different times of treatment Enzyme activity, with the treatment time of DTT, the enzyme activity of CBH1 decreased; with the increase of DTT treatment concentration, the enzyme activity of CBH1 decreased more rapidly. It can be seen from Figure 4 that the disulfide bond inside the CBH1 protein is very important for the maintenance of its enzymatic activity.

(2) Recovery of disulfide bond isomerase activity for inactivated CBH1

The pure protein of CBH1 was inactivated with 6M guanidine hydrochloride and 0.14M DTT, and the inactivated protein was purified with a desalting column. The inactivated CBH1 was treated with the TrPDI2 activity assay system, and the protein activity after treatment for 4, 8, 12, 16, 20, 24, 28, 32 and 36 hours was measured. As shown in Figure 5, the addition of TrPDI2 can partially restore the activity of the CBH1 protein that is completely reduced and inactivated (the enzyme activity of the reduced and inactivated CBH1 cannot be detected). loss of activity.

The two enzymes RNaseA and CBH1 involved in Example 2 and Example 3 all formed different numbers of disulfide bonds when forming the spatial structure, and their activity requires the correct formation of disulfide bonds, so they both belong to disulfide bonds. The disulfide bond isomerase TrPDI2 of the present invention has a certain effect on the recovery of the activities of these two enzymes, which proves that the TrPDI2 of the present invention can be used to stabilize and restore the activity of disulfide bond essential enzymes.

Although the embodiment of the present invention has been disclosed as above, it is not limited to the use listed in the specification and implementation, it can be applied to various fields suitable for the present invention, and it can be easily understood by those skilled in the art Therefore, the invention is not limited to the specific details and examples shown and described herein without departing from the general concept defined by the claims and their equivalents.

Claims (8)

1. a disulfide bond isomerase gene isolated from Trichoderma reesei, characterized in that its polynucleotide sequence is shown in (a) or (b): (a) the polynucleotide sequence shown in SEQ ID No.1; (b) A polynucleotide sequence complementary to the polynucleotide sequence in (a) according to the principle of complementary base pairing. 2. the isolated disulfide bond isomerase gene from Trichoderma reesei (Trichoderma reesei) as claimed in claim 1, is characterized in that, its polynucleotide sequence is also shown in (c): (c) The polynucleotide sequence shown in SEQ ID NO.1 (a) or the cDNA sequence of the polynucleotide sequence in (b): the polynucleotide sequence shown in SEQ ID NO.2. 3. A disulfide bond isomerase encoded by the disulfide bond isomerase gene isolated from Trichoderma reesei as claimed in claim 1. 4. disulfide bond isomerase as claimed in claim 3, is characterized in that, its amino acid sequence is the amino acid sequence shown in SEQ ID NO.3. 5. An expression vector containing the disulfide bond isomerase gene isolated from Trichoderma reesei as claimed in claim 1. 6. A recombinant host cell containing the expression vector of the disulfide bond isomerase gene according to claim 1. 7. The disulfide bond isomerase as claimed in claim 3, characterized in that it is used in increasing the expression of endogenous or exogenous proteins. 8. The disulfide bond isomerase as claimed in claim 3, characterized in that it is used in stabilizing and restoring disulfide bond essential enzyme activity.