Protein fusion tags and their applications

Abstract

The present invention belongs to the field of gene engineering technology, and specifically relates to a protein fusion tag and its application. The protein fusion tag includes a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2. The protein fusion tag can effectively increase the expression amount of the target protein and the water solubility of the target protein without affecting the activity of the target protein.

Description

Technical Field

[0001] The present invention belongs to the technical field of genetic engineering, and in particular relates to a protein fusion tag and an application thereof. Background Art

[0002] The E. coli expression system is currently the most commonly used recombinant protein expression system, and is widely used in the fields of medicine, industry, and molecular biology. In the pharmaceutical field, E. coli is used to produce small recombinant antibody fragments (scFv, Fab, and bispecific antibodies); in the industrial field, it is mainly used to produce industrial enzymes such as aspartase and peroxidase; in the field of molecular biology, it is used to prepare enzyme-linked immunosorbent assay (ELISA) kits, etc. As a prokaryotic organism, E. coli has the advantages of simple nutritional requirements, short passage time, and simple genetic manipulation. Therefore, when the E. coli expression system is used to express foreign proteins, its production cost is low and foreign proteins can be effectively expressed.

[0003] However, not every exogenous gene can be effectively expressed in E. coli, and the prokaryotic expression system also has defects in expressing heterologous proteins, such as: codon preference causes some genes with many rare codons not to be expressed in prokaryotic hosts; exogenous proteins are easily hydrolyzed in cells; protein misfolding causes exogenous proteins to aggregate in the cytoplasm in the form of biologically inactive inclusion bodies, etc. The use of protein fusion tags to help exogenous gene expression is currently the main solution to the inclusion body problem. At present, common fusion tags include: maltose binding protein (MBP), glutathione transferase (GST) and small molecule ubiquitin modified protein (SUMO); different fusion tags have advantages, but also certain limitations. There is no universal fusion tag that can solve the solubility problem of all proteins.

[0004] In summary, it is necessary to find a new and more effective fusion tag to help the large-scale expression of foreign proteins and improve solubility. Summary of the invention

[0005] In view of the above problems, one of the objectives of the present invention is to provide a protein fusion tag that can improve the expression and solubility of the target protein without affecting the activity of the target protein.

[0006] In order to achieve the above objectives, the following solutions can be adopted:

[0007] In one aspect, the present invention provides a protein fusion tag, which comprises a sequence as shown in SEQ ID NO.1 or SEQ ID NO.2.

[0008] Another aspect of the present invention provides a coding gene that expresses the above-mentioned protein fusion tag.

[0009] In another aspect, the present invention provides a recombinant vector comprising the above-mentioned encoding gene.

[0010] In another aspect, the present invention provides a recombinant bacterium, which comprises the above-mentioned protein fusion tag or the above-mentioned recombinant vector.

[0011] In another aspect, the present invention provides a transgenic cell line, which comprises the above-mentioned protein fusion tag or the above-mentioned recombinant vector or the above-mentioned recombinant bacteria.

[0012] In yet another aspect, the present invention provides an expression cassette, which includes the above-mentioned protein fusion tag or the above-mentioned recombinant vector or the above-mentioned recombinant bacteria or the above-mentioned transgenic cell line.

[0013] In another aspect, the present invention provides an application of the above-mentioned protein fusion tag in constructing a fusion protein expression system.

[0014] The beneficial effects of the present invention include at least: the protein fusion tag provided by the present invention can effectively increase the expression level of the target protein and improve the water solubility of the target protein without affecting the activity of the target protein. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 Schematic diagram for the construction of pNLD fusion tag vector;

[0016] Figure 2 SDS-PAGE electrophoresis detection of target proteins EcFabG and eGFP;

[0017] Figure 3 Grayscale analysis of target proteins EcFabG and eGFP;

[0018] Figure 4 is the catalytic activity of EcFabG towards C8-ACP;

[0019] Figure 5 To detect the activity of EcFabG on C8-ACP substrate;

[0020] Figure 6 Detection of the fluorescence intensity of eGFP;

[0021] in, Figure 2 In the figure, (a): expression analysis of LD-EcFabG fusion protein; (b): expression analysis of LD-eGFP fusion protein; S: supernatant of protein expression, P: precipitate after protein expression; from left to right: pET-28(b)-EcfabG, pNLD-EcfabG, pET-28(b)-egfp, pNLD-egfp; Figure 3In the figure, (a): grayscale analysis of LD-EcFabG and EcFabG; (b): grayscale analysis of LD-eGFP and eGFP; from left to right: grayscale values ​​of target proteins and SRP / EP ratios of pET-28(b)-EcfabG, pNLD-EcfabG, pET-28(b)-egfp, and pNLD-egfp; Figure 4 In the figure, 1: unlabeled fatty acid reaction with C8-ACP as substrate; 2: LD-labeled fatty acid reaction with C8-ACP as substrate; Figure 6 In the figure, from left to right are eGFP protein, tagged LD-eGFP protein, and eGFP protein after the fusion tag LD was cleaved by TEV enzyme. DETAILED DESCRIPTION

[0022] The examples are provided to better illustrate the present invention, but the present invention is not limited to the examples. Therefore, those skilled in the art may make non-essential improvements and adjustments to the implementation scheme according to the above invention content, which still fall within the protection scope of the present invention.

[0023] The terms used herein are only used to describe specific embodiments and are not intended to limit the present disclosure. Unless the context has a significantly different meaning, expressions in the singular include expressions in the plural. As used herein, it should be understood that terms such as "include", "have", "include" are intended to indicate the presence of features, numbers, operations, components, parts, elements, materials or combinations. The terms of the present invention are disclosed in the specification, and are not intended to exclude the possibility that one or more other features, numbers, operations, components, parts, elements, materials or combinations thereof may exist or may be added. As used herein, " / " may be interpreted as "and" or "or", depending on the circumstances.

[0024] On one hand, the present invention provides a protein fusion tag, which includes a sequence as shown in SEQ ID NO. 1 or SEQ ID NO. 2. It solves the problem that most proteins are difficult to express and insoluble in the E. coli expression system, and can effectively increase the expression amount of the target protein in the process of expressing the target protein in large quantities in E. coli, and improve the solubility of the target protein.

[0025] Specifically, the sequence shown in SEQ ID NO.1 is the amino acid sequence after transcription and translation of the alanine dehydrogenase E2 domain in the Escherichia coli K-12 genome (NCBI Protein ID: NP_414657.1), consisting of 630 amino acids; the predicted molecular weight of the protein is 66096.07 Daltons and the isoelectric point is 5.09; the sequence shown in SEQ ID NO.2 is a derived amino acid obtained by recombining amino acids 1-34 and 239-290 in the sequence shown in SEQ ID NO.1.

[0026] Another aspect of the present invention provides a coding gene that expresses the above-mentioned protein fusion tag. Specifically, the above-mentioned protein fusion tag is applied to the expression of the target protein, and the coding gene of the protein fusion tag works together with the coding gene of the target protein.

[0027] Further, in some embodiments, the above-mentioned coding gene may include a sequence as shown in SEQ ID NO.3 or SEQ ID NO.4. Specifically, the sequence shown in SEQ ID NO.3 is the alanine dehydrogenase E2 domain sequence in the Escherichia coli K-12 genome (NCBI Gene ID: 944794), which consists of 1893 nucleotides, that is, it encodes the sequence shown in SEQ ID NO.1; the sequence shown in SEQ ID NO.4 is the sequence shown in SEQ ID NO.3 with one or several nucleotides replaced, and the amino acid expressed by it has more than 90% homology with the sequence shown in SEQ ID NO.1.

[0028] On the other hand, the present invention provides a recombinant vector, which includes the above-mentioned coding gene. Specifically, the above-mentioned coding gene and the target gene are inserted into an expression vector to form a recombinant vector for the expression of the target protein, which can increase the expression amount of the target protein and improve the solubility. In some specific implementations of the present invention, 3-ketoacyl ACP reductase (FabG) and enhanced green fluorescent protein (eGFP) are used as target proteins for experimental demonstration, which increases the expression amount and solubility of the target protein, and the activity of the target protein is not affected by the above-mentioned protein fusion tag. It should be understood that the target gene is not only applicable to the above-mentioned 3-ketoacyl ACP reductase (FabG) and enhanced green fluorescent protein (eGFP).

[0029] Furthermore, in some embodiments, the above recombinant vector may also include an expression vector pET-28b(+). Specifically, the expression vector in the above recombinant vector may be a vector known in the art, such as the expression vector pET-28b(+).

[0030] Specifically, the construction method of the above-mentioned recombinant vector can be carried out according to the following steps: the above-mentioned protein fusion tag gene and the nucleotide sequence with the TEV restriction site are connected through a small linker, and inserted between the NcoI and BamHI restriction sites of the expression vector to form the above-mentioned recombinant expression vector; after the expression of the target protein is completed, the fusion tag can be cleaved by TEV protease, which can effectively increase the expression amount of the target protein without affecting the spatial structure and subsequent activity of the target protein, thereby obtaining a tag-free target protein and reducing subsequent downstream costs.

[0031] In another aspect, the present invention provides a recombinant bacterium, which includes the above-mentioned protein fusion tag or the above-mentioned recombinant vector. Specifically, the target protein generally selects some engineering bacteria for the expression of the target protein, and the engineering bacteria are bacteria known in the art, and Escherichia coli is a commonly used target protein expression engineering bacteria in the art.

[0032] In another aspect, the present invention provides a transgenic cell line, which includes the above-mentioned protein fusion tag or the above-mentioned recombinant vector or the above-mentioned recombinant bacteria. Specifically, the expression of the target protein is usually also achieved by using some engineering cells, and the above-mentioned protein fusion tag is combined with the target gene in the engineering cells for expression.

[0033] In another aspect, the present invention provides an expression cassette, which includes the above-mentioned protein fusion tag or the above-mentioned recombinant vector or the above-mentioned recombinant bacteria or the above-mentioned transgenic cell line. Specifically, the above-mentioned protein fusion tag assists the target protein encoding gene usually with the help of some vectors to facilitate operation and transportation, such as through the expression cassette, which is a conventional expression cassette in the art.

[0034] In another aspect, the present invention provides an application of the above-mentioned protein fusion tag in constructing a fusion protein expression system.

[0035] The purpose of the present invention can be achieved by the following technical solutions:

[0036] The present invention provides a novel protein fusion tag for increasing the expression and solubility of a target protein in Escherichia coli.

[0037] In order to better understand the present invention, the content of the present invention is further explained below in conjunction with specific examples, but the content of the present invention is not limited to the following examples.

[0038] In the following examples, the Escherichia coli MG1655, DH5α, BL21 (DE3), and plasmid pET-28b (+) used were deposited by the applicant; reagents such as restriction endonucleases and ligases were purchased from Bio-Rad Biotechnology (Beijing) Co., Ltd.; and PCR primers were synthesized by Guangzhou Branch of Sangon Biotechnology (Shanghai) Co., Ltd.

[0039] Example 1 Preparation of fusion tag vector

[0040] In the implementation of the present invention, according to Figure 1 The schematic diagram of constructing the pNLD fusion tag vector shown in the figure is used to construct the pNLD fusion tag vector, as follows:

[0041] (1) Primer design

[0042] According to the LD gene, EcfabG gene, egfp gene and upstream and downstream sequences, primers were designed (the underlined parts are restriction sites), as shown in Table 1 below.

[0043] Table 1 Primers designed in Example 1

[0044]

[0045]

[0046] (2) Construction of pNLD vector, experimental group expression vector, and control group vector

[0047] When designing primers, the present invention pre-designs short overlapping extension connectors at the ends of primers P2, P3, P4, P5, and P6, and uses overlapping extension PCR method with P1 as the upstream primer to splice the downstream fragments;

[0048] PCR technology was used to amplify the LD tag gene (shown in SEQ ID NO. 4) with P1 and P2 primers to obtain a 258 bp LD gene DNA fragment (50 μl amplification system: 1 μl total DNA, 2 μl of each P1 / P2 primer, 45 μl of 1.1×MixGreen; amplification conditions: 98°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 57°C annealing for 10 s, 72°C extension for 10 s; program cycle number: 35 cycles);

[0049] Using the LD gene DNA fragment as a template, amplification was performed using P1 and P3 primers designed to include the KpnI site to the linker fragment to obtain a 305 bp LD-linker fragment (50 μl amplification system: 1 μl of P1P2 fragment, 2 μl of P1 / P3 primers, 45 μl of 1.1×MixGreen; amplification conditions: 98°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 57°C annealing for 10 s, 72°C extension for 10 s; program cycle number: 35 cycles);

[0050] Then, the LD-linker fragment was used as a template, and P1 and P4 primers designed to contain TEV protease cleavage sites were used for amplification to obtain a 326 bp LD-linker-TEV fragment (50 μl amplification system: 1 μl of P1P3 fragment, 2 μl of P1 / P4 primers, 45 μl of 1.1×MixGreen; amplification conditions: 98°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 57°C annealing for 10 s, 72°C extension for 10 s; program cycle number: 35 cycles);

[0051] Then, the LD-linker-TEV fragment was used as a template, and P1 and P5 primers designed to contain the hexahistidine coding sequence were used for amplification to obtain a 342 bp LD-linker-TEV-His fragment (50 μl amplification system: 1 μl of P1P4 fragment, 2 μl of P1 / P5 primers, 45 μl of 1.1×MixGreen; amplification conditions: 98°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 57°C annealing for 10 s, 72°C extension for 10 s; program cycle number: 35 cycles);

[0052] Then, P1 and P6 primers designed to contain BamHI sites were used for amplification to obtain a complete LD-linker-TEV-His fragment of 355 bp containing NcoI and BamHI sites (50 μl amplification system: 1 μl of P1P5 fragment, 2 μl of P1 / P6 primers, 45 μl of 1.1×MixGreen; amplification conditions: 98°C pre-denaturation for 2 min, 98°C denaturation for 10 s, 57°C annealing for 10 s, 72°C extension for 10 s; program cycle number: 35 cycles);

[0053] The RBS region of the pET-28b(+) vector sequence was transformed into the MCS region. First, the pET-28b(+) was digested with restriction endonucleases NcoI and BamHI (enzyme digestion conditions were: 60 μl of fragment or plasmid, 10 μl of 10× enzyme digestion buffer, 2 μl of BamHI, 2 μl of NcoI, and double distilled water to 100 μl, and reacted at 37°C for 4 h) to obtain a large fragment of 5271 bp;

[0054] The LD-linker-TEV-His fragment was digested with NcoI and BamHI, and connected to the pET-28b(+) vector that was also double-digested (the connection conditions were: 10 μl of the digested fragment, 3 μl of the digested vector, 2 μl of 10× ligase buffer, 0.5 μl of T4 DNA ligase, and double-distilled water to 20 μl; connection at 16°C for 8 h) to obtain the fusion tag vector pNLD, which was then heat-shock transformed (the transformation conditions were: 20 μl of the connection system, 100 μl of competent cells, 30 min of ice bath, 90 s of heat shock, 30 min of ice bath, and 700 μl of LB culture medium for 45 min), and colony PCR screening (10 μl amplification system: 1 μl of the recombinant bacterial solution, 0.5 μl of each of the upstream and downstream primers of pET-T7, 2× DNA polymerase Taq Mix 10 μl, add double distilled water to 20 μl; amplification conditions: 95℃ pre-denaturation for 5 min, 95℃ denaturation for 30 s, 53℃ annealing for 30 s, 72℃ extension for 1 min; program cycle number: 30 cycles), screen the positive recombinants and send them to the biological company for sequencing verification;

[0055] After sequencing was correct, the recombinant plasmid pNLD was extracted (plasmid extraction kit was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B110091-0100), and the restriction endonucleases NdeI and SalI were digested and purified for later use; pET-28b(+) was then extracted and digested with NdeI and SalI for later use (gel recovery kit was purchased from Sangon Biotech (Shanghai) Co., Ltd., catalog number B110092-0100); linearized pNLD plasmid and pET-28b(+) plasmid were obtained.

[0056] Using the total DNA of Escherichia coli MG1655 as a template, the upstream and downstream primers P7 and P8 of the 3-ketoacyl ACP reductase gene were used for amplification to obtain a 738bp EcfabG gene fragment (50μl amplification system: 1μl total DNA, 2μl of each P7 / P8 primer, 45μl of 1.1×MixGreen; amplification conditions: 98℃ pre-denaturation for 2min, 98℃ denaturation for 10s, 57℃ annealing for 10s, 72℃ extension for 10s; program cycle number: 35 cycles);

[0057] Using pET-egfp plasmid as template, the upstream and downstream primers P9 and P10 of enhanced green fluorescent protein gene were used for amplification to obtain 717bp of egfp gene fragment (50μl amplification system: 1μl of plasmid DNA, 2μl of P9 / P10 primers, 45μl of 1.1×MixGreen; amplification conditions: 98℃ pre-denaturation for 2min, 98℃ denaturation for 10s, 57℃ annealing for 10s, 72℃ extension for 10s; program cycle number: 35 cycles);

[0058] The EcfabG and egfp fragments were digested with NdeI and SalI and ligated to the pET-28b(+) vector that had been digested with the same double enzymes (the ligation conditions were: 10 μl of the digested fragment, 3 μl of the digested vector, 2 μl of 10× ligase buffer, 0.5 μl of T4 DNA ligase, and double distilled water to 20 μl. The ligation was carried out at 16°C for 8 h to obtain the fusion tag vectors pNLD-EcfabG, pNLD-egfp, pET-EcfabG, and pET-egfp.

[0059] Example 2 Escherichia coli protein expression and analysis

[0060] After sequencing, the pNLD-EcfabG, pNLD-egfp, pET-EcfabG, and pET-egfp plasmids in Example 1 were transformed into Escherichia coli BL21 (DE3) strains, and then single colonies were picked and cultured in LB liquid medium containing Kan antibiotic (Kan 30 mg / ml) at 37° C. overnight, which was called seed liquid;

[0061] Take 100 μl of seed solution and add it to 5 ml of LB liquid medium, add 5 μl of Kan, and culture at 37°C with shaking at 200 rpm until the bacterial solution OD reaches 0. 600 At about 0.8, add 5 μL of isopropylthio-β-D-galactoside (IPTG 200 mg / ml) and continue to culture for 4 hours; then centrifuge at 4000 rpm for 10 minutes to collect the bacteria, discard the supernatant, and collect the bacterial precipitate; resuspend the precipitate with 1 ml of lysis buffer, and crush the suspension in an ice bath with ultrasonic waves for 5 minutes. After the end, centrifuge at 12000 rpm for 10 minutes to collect the supernatant (S) and precipitate (P) respectively; when inducing expression, the experimental group and the control group need to be induced under the same conditions;

[0062] The supernatant and precipitate of the experimental group and the control group were subjected to SDS-PAGE detection to analyze the expression product. The results of SDS-PAGE are as follows: Figure 2 As shown, the size of the target protein is consistent with expectations; the expression amount and solubility ratio of the target protein with the expression vector containing the LD fusion tag are greatly improved;

[0063] SDS-PAGE grayscale analysis was performed based on bovine serum albumin (BSA). Figure 3As shown, based on the BSA concentration of 2 mg / ml, it was calculated that the target protein pET-28(b)-EcfabG co-expressed 4.26 μg, pNLD-EcfabG co-expressed 9.01 μg, pET-28(b)-egfp co-expressed 15.16 μg, and pNLD-egfp co-expressed 39.38 μg; that is, the vector with LD fusion tag can increase the expression amount by 2.11-2.59 times when expressing the target protein; at the same time, when expressing eGFP protein, the solubility of the pNLD-eGFP vector to the target protein is increased by 1.61 times compared with the pET-28(b)-egfp vector.

[0064] Example 3 Detection of EcFabG and eGFP protein activities

[0065] In the embodiment of the present invention, the activity detection of EcFabG refers to the following method: 3-ketoacyl ACP reductase (FabG) catalyzes the reduction of 3-ketoacyl ACP to 3-hydroxyacyl ACP, and is one of the key enzymes in bacterial fatty acid synthesis reaction; EcFabG is expressed and purified, and the activity is measured; 3-ketodecanoyl ACP is used as a substrate to measure the activity of EcFabG in oxidizing NADPH: the reaction volume is 100 μl, which contains 0.1M sodium phosphate (pH 7.4); 5mM dithiothreitol; 200μM NADPH; 200μM malonyl CoA; 1μg octanoyl ACP; 1μg holo-ACP; 0.2μg FabD and 0.2μg FabB; after adding 20μM Tag-FabG, the decrease rate of absorbance at 340nm is measured at 25°C using a UV-visible spectrophotometer;

[0066] In the embodiment of the present invention, the eGFP activity detection refers to the following method: taking the untagged eGFP protein as the control, taking the same concentration of protein, diluting it to 1pM and adding it to a 1ml quartz cuvette, and performing fluorescence luminescence detection in a fluorescence spectrophotometer, the maximum excitation wavelength used is 489nm, the maximum emission wavelength is 511nm, and the fluorescence intensity of the eGFP protein with the tag of the present invention is compared at the absorption peak;

[0067] (1) Analysis of EcFabG protein activity

[0068] After mixing 10 mM octanoic acid-ACP and malonyl-ACP, 0.2 μg FabB, 0.2 μg FabA, 0.2 μg FabI and 0.2 μg FabG were added, and the mixture was reacted at 37°C for 1 hour, and electrophoresed using 0.5 M urea No SDS-PAGE. The results are shown in Figure 4 As shown, the formation of decanoic acid can proceed normally; the reaction with octanoic acid-ACP as substrate is carried out, the result is Figure 5As shown, the activity of FabG protein was not affected with LD tag;

[0069] (2) eGFP protein activity analysis

[0070] The protein solution diluted to 1pM was added to a 1mL fully transparent quartz dish. At an excitation wavelength of 489nm and an emission wavelength of 511nm, the fluorescence intensity of the tagged eGFP and the eGFP protein after TEV cleavage did not change significantly. The results are as follows: Figure 6 shown.

[0071] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the purpose and scope of the technical solution of the present invention, which should be covered by the scope of the claims of the present invention.

Claims

1. The application of protein fusion tag in constructing fusion protein expression system is characterized by: The sequence of the protein fusion tag is shown in SEQ ID NO.2.

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