WO2017075863A1 - Fused protein expression vector of chaperone-like protein - Google Patents

Abstract

A fused protein expression vector of a chaperone-like protein. The cloning region upstream of the expression vector comprises a nucleotide sequence encoding a human free fatty acid binding protein. The nucleotide sequence downstream preferably comprises a flexible joint region and a multi-cloning region for insertion of a target protein encoding nucleic acid.

Description

Fusion protein expression vector of a chaperon-like protein Technical field

The invention relates to a fusion protein expression technology, in particular to a fusion protein expression vector of a chaperone protein, belonging to the field of genetic engineering and protein expression engineering.

Background technique

The function of a protein depends on the natural conformation of the protein. It is generally believed that the tertiary and quaternary structure of the protein molecule is entirely determined by the amino acid sequence of the polypeptide. However, some studies in recent years have shown that many proteins are involved in the folding and assembly of other proteins or enzymes, of which molecular chaperones are the most studied. Molecular chaperones are a class of evolutionarily very conserved proteins that bind non-specifically to polypeptide chains that differ in structure, size, location, and final function. They catalyze the formation of specific conformations of proteins and participate in the folding, assembly, and transport of proteins in vivo. .

The concept of molecular chaperones was first proposed by Laskey et al. (1978). They discovered an acidic nuclear protein, Nucleoplasmin, when studying the formation of Xenopus nucleosomes. Experiments have shown that it is necessary for the assembly of DNA and histones into nucleosomes. At physiological ionic strength, DNA is mixed with histones in vitro and cannot self-assemble, but forms a precipitate. If the histone is mixed with excess Nucleoplasmin and then DNA is added, a nucleosome structure can be formed, and there is no Nucleoplasmin in the resulting nucleosome. It is now believed that the role of Nucleoplasmin may be to avoid the formation of non-specifically bound insoluble polymers by strong electrostatic attraction between negatively charged DNA and positively charged histones.

Molecular chaperone is an evolutionarily very conserved protein superfamily that is widely distributed in various organisms and was first identified as HSP (heat shock protein). Newly synthesized polypeptide chains must first be folded and assembled to form a specific three-dimensional structure to be active. In the process of folding the polypeptide chain, abnormal proteins are often produced to form aggregates, and in the genetic engineering protein expression system, it is called inclusion. Protein molecules with a chaperone-like effect can effectively regulate the correct folding of other polypeptide chains, thereby avoiding the formation of inclusion bodies.

In 1993, E11is made a more precise definition of molecular chaperones: molecular chaperones are a class of proteins that are related to each other and are capable of binding and stabilizing the unstable conformation of another protein. Their function is to help other peptides. Structured substances undergo correct non-covalent assembly in vivo, controlled binding and release, promotion of folding of nascent polypeptides, assembly or degradation of multimers, and transmembrane transport of organelle proteins, and are not assembled proteins. A component of its normal biological function ^[3] . Proteins (also known as accessory proteins) that aid in the folding of nascent peptides are known to have at least three broad categories: one is a universal molecular chaperone that helps correct folding, prevents and corrects incorrect folding. The other type is a molecular chaperone with enzyme activity, also known as a folding enzyme. There are two folding enzymes: one is protein disulfide isomerase (PDI); the other is peptidylprodylcis-trans isomerase (PPI). At the time, Ellis made it clear that PDI is not a molecular chaperone ^[3] , and it has been shown that these two isomerases are both enzymes and molecular chaperones ^[4] . The third category is intramolecular chaperones. Some studies have shown that many proteins that are synthesized in the precursor form of the leader peptide (Pro peptide) must be folded and matured to have the presence of a Pro peptide, and do not fully comply with the Anfinsen rule. Such leader peptides are called intramolecular chaperones (IMCs).

Recently, Dong Xiaoyan et al. used the immobilized molecular chaperone GroE to study its renaturation effect on denatured lysozyme and solved the problem of molecular chaperone reuse. Teshima et al. assisted the folding renaturation of amylase, carbonic anhydrase, and DNase in vitro using immobilized molecular chaperones. It has also been reported that renaturation of target proteins by "small molecular chaperones" can effectively promote the renaturation of cyclophilin A, rhodanese and bacillus RNase.

Bacterial glutathione S-transferase (GST) is a kind of self-folding protein. It was first used by Pharmacia to construct pGEX series prokaryotic expression vector. Due to its high expression and self-folding ability, GST has many The concept of a "companion-like protein" protein emerges from the high expression of many exogenous proteins fused downstream or by increased soluble expression.

NEB uses maltose binding protein to construct pMAL series prokaryotic expression vector, which can also promote the soluble expression of partial fusion protein, has chaperone-like protein characteristics, and further utilizes the affinity of MBP for maltose to achieve amylose. The (Amylose) column was affinity purified for the fusion protein.

Novagen's Disulfide bond formation protein A (DsbA) is a folding enzyme that is involved in the formation of disulfide bonds in the process of nascent protein folding in the periplasmic cavity of E. coli. Prokaryotic expression vectors can contribute to increased solubility of the expressed exogenous protein. DsbA belongs to a chaperone protein.

Takara Bio INC. constructed and sold a series of co-transfected prokaryotic expression vectors containing cpn60, in an attempt Improve the renaturation of the expressed rim protein and achieved good results. Previous studies have shown that GroEL can significantly improve the refolding efficiency of genetically engineered protein scorpion Cn5, cyclophilin A and 吲哚3-glycerol phosphate synthase in vitro.

Small ubiquitin-like modified proteins are widely found in eukaryotes and are a class of small-molecule polypeptides involved in post-translational modification of proteins. They have good self-folding properties during E. coli expression and also induce some fusions downstream. The folding action of proteins is classified as a chaperon-like protein. Such prokaryotic expression vectors containing SUMO have been widely used in foreign countries.

Summary of the invention

In view of the above findings, it is an object of the present invention to provide a novel fusion protein expression vector which functions like a chaperone-like protein. It also uses human own protein, which is characterized by non-rejection or weak immunogenicity.

The invention is achieved by the following technical solutions:

Use of the human free fatty acid binding protein of any one of SEQ ID NOS: 1 to 9 in the construction of a fusion protein expression vector that functions like a chaperon-like protein.

For the application, preferably the optimized human free fatty acid binding protein coding sequence is inserted into the fusion protein expression vector as an upstream protein.

A fusion protein expression vector for a chaperon-like protein, comprising a nucleic acid sequence encoding a human free fatty acid binding protein upstream of a cloning region thereof.

The fusion protein expression vector, downstream of the human free fatty acid binding protein, preferably comprises a flexible linker region and a polyclonal region for insertion of the protein of interest.

Wherein the nucleic acid sequence encoding the human free fatty acid binding protein is a codon-optimized coding sequence, the homology of the encoded human free fatty acid binding protein and the homology of the human natural free fatty acid binding protein is equal to or greater than 85 %.

The nucleic acid sequence encoding the human free fatty acid binding protein is further preferably as shown in SEQ ID NO: 10.

The flexible linker region and the polyclonal region sequence located downstream of the nucleic acid sequence encoding the human free fatty acid binding protein in the expression vector are preferably as set forth in SEQ ID NO: 13.

The upstream of the nucleic acid sequence encoding the human free fatty acid binding protein in the expression vector preferably further comprises a sequence encoding a tag for isolating and purifying the fusion protein, and further preferably a sequence encoding a histidine tag.

The use of the expression vector of the present invention for expressing a protein of interest.

A method for expressing a fusion protein of a chaperone-like protein, which comprises inserting a coding sequence of a target protein into a polyclonal region downstream of the flexible linker region of the expression vector according to any one of claims 3 to 8 to obtain a recombinant expression vector containing the coding sequence of the fusion protein. The vector is transfected into a host cell, and the host cell is cultured to express the fusion protein.

Beneficial effects:

1. The recombinant expression vector constructed by the present invention is capable of promoting or inducing folding of a fused downstream protein, having the properties of a protein chaperone-like protein;

2. The FABP protein is human-derived, and theoretically, it has no immunogenicity to the human body, and is suitable for solving the problem of target protein inclusion bodies in the pharmaceutical industry, and can retain the fusion protein together.

DRAWINGS

Figure 1. Schematic diagram of hFABP fusion protein

Figure 2. Physical map of pET28a-hFABP6 expression vector

Figure 3. Physical map of pET28-hFABP-hEGF fusion protein expression vector

Figure 4. Physical map of pET28-hEGF expression vector

detailed description

The application of the present invention will now be described by way of specific examples, which are merely illustrative of the application of the present invention, which will help to better understand the application of the present invention, but do not limit the scope of application of the present invention. The experimental methods described in the following examples are conventional methods unless otherwise specified; the reagents and materials are commercially available unless otherwise specified.

Example 1

A method for constructing a fusion protein expression vector of a chaperon-like protein, comprising the steps of:

Step 1. Artificial synthesis and optimization of the coding DNA of hFABP. In this example, hFABP6 is taken as an example (SEQ ID NO: 6):

(1) The hFBP6 amino acid sequence was reverse translated into a DNA coding sequence, and the sequence was obtained by codon preference and ribosome binding region sequence optimization (SEQ ID NO: 10), but not limited to this sequence, and the translated protein amino acid sequence was the same. The source is equal to or greater than 85%.

(2) The coding sequence was segmented into oligonucleotide single-stranded DNA (SEQ ID NOS: 11 to 24).

(3) Polymerase chain reaction (PCR) method to synthesize a full-length coding DNA sequence comprising 5'-6xHisTag (SEQ ID NO: 25, corresponding amino acid sequence is SEQ ID NO: 26), hFBP6 amino acid coding sequence, 3'- The flexible linker sequence (Linker) and the polyclonal region (SEQ ID NO: 27, corresponding amino acid sequence is SEQ ID NO: 28).

(4) The full-length DNA sequence (SEQ ID NO: 29, the encoded amino acid sequence is shown in SEQ ID NO: 30) was verified by a sequencing reaction.

Step 2, carrier preparation:

(1) The hFABP6 coding sequence (SEQ ID NO: 29) synthesized in the first step, comprising a 5'-6xHisTag, a 3'-flexible linker sequence (Linker) and a polyclonal region, which are digested with restriction endonucleases, The agarose gel was purified to obtain an insert of a viscous linker with Nco I and Xho I, respectively.

(2) The expression vector pET28a is prepared, but is not limited to the expression vector:

1 1 μg of pET28a vector was taken and digested with restriction endonucleases Nco I and Xho I.

2 The agarose gel electrophoresis was used to separate and purify the linearized pET28a vector.

3 The insert containing the hFABP6 sequence was ligated with the pET28a expression vector prepared in step 2 using T4 DNA ligase.

4 The E. coli competent strain DH5α was transformed, and the culture dish containing kanamycin antibiotic was cultured overnight, then single colonies were picked, positive clones were identified by PCR, and DNA plasmids were routinely prepared.

5 The positive clone plasmid was sent to DNA sequence analysis, and the clone of the correct sequence was selected and retained for expression test.

6 vector named pET28a-hFABP6 (SEQ ID NO: 31)

Step 3: Expression test of the carrier:

(1) The pET28a-hFABP6 vector DNA plasmid obtained in the second step was transformed into E. coli BL21 (DE3) competent cells to obtain Kan-resistant colonies.

(2) Pick a few single colonies, culture in LB medium, induce IPTG for 4-12 hours, collect 1 mL of bacterial solution, collect the cells by centrifugation, wash once in 1×PBS, and resuspend in 0.5 mL of 1×PBS, sonicated bacteria. Body, centrifuge to precipitate.

(3) Take an appropriate amount of 20 μL of the supernatant and mix it with the loading buffer, heat denature at 95 ° C for 10 minutes, collect by centrifugation to the bottom of the tube, onto ice.

(4) 10 μL of 15% polyacrylamide was applied for electrophoresis, Coomassie blue staining and decolorization, and the expression of protein bands and hFABP6 protein were observed.

(5) Recombinant hFABP6, including N-HisTag, flexible linker, and polyclonal region, totaling 158 aa, molecular weight 17.2 kDa. hFABP6 accounts for 20% of total bacterial proteins and is soluble in expression.

Example 2

Construction of pET28-hFABP-hEGF fusion protein expression vector

1. Construct hFABP-hEGF from pET28a-hFABP6:

1. A DNA sequence encoding a hEGF mature peptide (SEQ ID NO: 32);

2. PCR method introduces Kpn I (5'-end) and Xho I (3'-end) double-enclosure cloning sites;

Upstream primer: 5'-TTTTGGTACCAACTCTGACTCTGAATGCC-3' (SEQ ID NO: 33), downstream primer: 5'-TTTTCTCGAGTTAACGCAGCTCCCACCATTTGAG-3', (SEQ ID NO: 34)

3. Kpn I/Xho I double digestion treatment expression vector pET28a-hFABP6;

4. The hEGF-encoding DNA insert and the pET28a-hFABP6 vector fragment were separately recovered by gel;

5. The T4 DNA ligase is ligated to the pET28a-hFABP6 vector fragment and the hEGF insert DNA, and the recombinant expression vector comprises the histidine tag coding sequence represented by SEQ ID NO: 35 - hFABP6 protein coding sequence - flexible linker region - hEGF Insertion DNA, the corresponding amino acid sequence of which is shown in SEQ ID NO: 36;

6. The recombinant expression vector obtained in the previous step was directly transformed into E. coli BL21 (DE3) competent cells, and single colony PCR was used for identification. The PCR primers used were pET28a sequencing primers T7-primer (SEQ ID NO: 37) and T7-terminator. . (SEQ ID NO: 38)

7. A positive clone containing a larger band than the pET28a-hFABP6 empty vector PCR fragment was retained, and the DNA sequence analysis was performed, and the clone of the correct sequence was retained for test expression analysis.

8. The correct clone was named pET28-hFABP-hEGF (SEQ ID NO: 39).

2. Construction of control clone pET28-hEGF

1. Synthesizing a DNA sequence encoding a mature peptide of hEGF;

2. PCR method introduces Nco I (5'-end) and Xho I (3'-end) double restriction enzyme cloning site and initiation codon ATP; upstream primer: 5'-TTTTCCATGAACTCTGACTCTGAATGCC-3' (SEQ ID NO: 40), downstream primer: 5'-TTTTCTCGAGTTAACGCAGCTCCCACCATTTGAG-3' (SEQ ID NO: 34)

3. Nco I/Xho I double digestion treatment expression vector pET28a;

4. The hEGF-encoding DNA insert and the pET28a vector fragment are separately recovered by gel;

5. The T4 DNA ligase is ligated to the pET28a DNA fragment and the hEGF DNA fragment;

6. Directly transform E. coli BL21 (DE3) competent cells and select single colony PCR for identification. The PCR primers used were pET28a sequencing primers T7-Primer (SEQ ID NO: 37) and T7-Terminator. (SEQ ID NO: 38)

7. Preserve positive clones containing a larger band than the pET28a empty vector PCR fragment, send DNA sequence analysis, and retain the correct sequence clone for test expression analysis.

8. The correct clone was named pET28-hEGF (SEQ ID NO: 41).

Second, test expression analysis

1. Amplifying BL21(DE3) strain containing pET28-hFABP-hEGF and pET28-hEGF plasmid DNA, respectively;

2. IPTG induces protein expression;

3. Collect the cells, wash once with ×PBS, add 500 μL of 1×PBS, and disrupt the cells by ultrasonic wave;

4. Centrifuge the supernatant and centrifuge to add 500 μL of 1×PBS to 50 μL of the same amount of 2× Loading Buffer, denature at 95 °C for 10 minutes, load, and 20% polyacrylamide gel electrophoresis;

5. Coomassie blue staining and observation after decolorization.

6. It was found that the soluble expression of hFABP6-hEGF fusion protein expression accounted for 50%, while hEGF alone expressed all of them as inclusion bodies, indicating that the upstream protein hFABP6 has the effect of promoting hEGF folding.

7. Conclusion: In the pET28-hFABP6-hEGF fusion protein expression vector, the upstream protein hFABP6 has a protein chaperone-like effect that promotes hEGF folding.

Claims (10)

1. Use of the human free fatty acid binding protein of any one of SEQ ID NOS: 1 to 9 in the construction of a fusion protein expression vector that functions like a chaperon-like protein.
2. The use according to claim 1, characterized in that the optimized coding sequence of the human free fatty acid binding protein is inserted into the fusion protein expression vector as an upstream protein.
3. A fusion protein expression vector for a chaperon-like protein, characterized in that a nucleic acid sequence encoding a human free fatty acid binding protein is contained upstream of the cloning region.
4. The fusion protein expression vector according to claim 3, wherein the human free fatty acid binding protein comprises a flexible linker region downstream and a polyclonal region for insertion of the target protein.
5. The fusion protein expression vector according to claim 3, wherein the nucleic acid sequence encoding the human free fatty acid binding protein is a codon-optimized coding sequence, and the homology of the human free fatty acid binding protein encoded by the human The homology of the native free fatty acid binding protein is equal to or greater than 85%.
6. The fusion protein expression vector according to claim 5, wherein the nucleic acid sequence encoding the human free fatty acid binding protein is represented by SEQ ID NO: 10.
7. The fusion protein expression vector according to claim 4, wherein the flexible linker region and the polyclonal region sequence located downstream of the nucleic acid sequence encoding the human free fatty acid binding protein are shown in SEQ ID NO: 13.
8. The fusion protein expression vector according to claim 3, characterized in that the nucleic acid sequence encoding the human free fatty acid binding protein in the expression vector further comprises a sequence encoding a tag for isolating and purifying the fusion protein, preferably encoding a histamine. The sequence of the acid tag.
9. Use of the expression vector of any one of claims 3 to 8 for expression of a protein of interest.
10. A method for expressing a fusion protein of a chaperone-like protein, which comprises inserting a coding sequence of a protein of interest into a polyclonal region downstream of the flexible linker region of the expression vector according to any one of claims 3 to 8 to obtain a coding sequence containing the fusion protein. The recombinant expression vector is transfected into the host cell, and the host cell is cultured to express the fusion protein.

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