CN116041451B - Intein variant and application thereof in preparation of blue copper peptide by biological method - Google Patents

Abstract

The invention discloses an intein variant and application thereof in preparing blue copper peptide by a biological method, wherein the amino acid sequence of the intein variant is shown as SEQ ID NO. 1, and the nucleotide sequence of the intein variant is shown as SEQ ID NO. 2. The recombinant expression vector of the blue copper peptide with high efficiency and stability is prepared by forming fusion protein with GHK and introducing the fusion protein into a vector plasmid, and the blue copper peptide with high purity can be obtained by induction expression. Moreover, the blue copper peptide based on the method has low preparation difficulty, can effectively reduce pollution and the generation of toxic and harmful byproducts, has large output, is suitable for large-scale industrialized mass production of the blue copper peptide, and has great market value.

Description

Intein variant and application thereof in preparation of blue copper peptide by biological method

Technical Field

The invention belongs to the field of molecular biology, and particularly relates to an intein variant and application thereof in preparing blue copper peptide by a biological method.

Background

Blue copper peptide, also known as tripeptide-1 copper, is a tripeptide-coupled copper ion consisting of glycine Gly (G) -histidine His (H) -lysine Lys (K) residues to form an oligopeptide-copper complex (GHK-Cu). GHK is a relatively rare amino acid sequence in human protein, so that GHK short peptide generated by self-degradation of the body has extremely low content in the body. The concentration of blue copper peptide in plasma was about 200 μg/mL at age 20, and gradually decreased with age to 80 μg/mL at age 60. Blue copper peptides play important biological functions in the body, including promoting collagen and elastin regeneration, promoting wound healing, anti-inflammation, repairing DNA damage caused by ultraviolet, and the like. Therefore, the blue copper peptide has extremely high application value in the medical and skin care fields and also has extremely high market demand.

The blue copper peptide sold in the market at present mainly comes from chemical synthesis modes, including liquid phase synthesis and solid phase synthesis, and is obtained by gradually mixing and adding amino acid residues one by one to obtain tripeptide, and then coupling with copper ions. These methods can obtain blue copper peptides, but the yield is extremely low. This is mainly because chemical synthesis brings about a lot of synthesis byproducts, and some byproducts have even large cytotoxicity, so that the production and purification of the blue copper peptide consume a lot of cost, and serious pollution is easy to bring about, which greatly limits the application of the blue copper peptide. Therefore, development of a more green, economical and efficient synthesis mode is urgently needed for industrial production of blue copper peptides.

Disclosure of Invention

The present invention aims to solve at least one of the technical problems in the prior art described above. Therefore, the invention provides an intein variant and application thereof in preparing blue copper peptide by a biological method, and the blue copper peptide can be efficiently and massively produced by fusing the intein variant and GHK tripeptide, thereby effectively avoiding the production of byproducts and toxic and harmful substances, and the obtained blue copper peptide has excellent quality and high purity and is far superior to the existing traditional blue copper peptide production method.

In a first aspect of the invention there is provided an intein variant resulting from mutation of the G14 gene fragment of Chitinophagaceae bacterium.

According to a first aspect of the invention, in some embodiments of the invention, the gene fragment is a fragment of amino acid residues 303 to 431 of MCF 8340111.1.

The inventors found that the G14 gene of Chitinophagaceae bacterium (database number: MCF 8340111.1) has some homology to the sequence of the conventional commercial intein Ssp dnaB, and that three-level results of MCF8340111.1 and Ssp dnaB were predicted based on alpha Fold2, and that both were highly similar in three-level structure, it was hypothesized that the fragment in MCF8340111.1 might function as an intein similar to Ssp dnaB. Based on this, it was further found that the fragment of amino acid residues 303 to 431 of MCF8340111.1 can be used as a mutant parent.

In some embodiments of the invention, the amino acid sequence of the intein variant is shown in SEQ ID NO. 1.

In some embodiments of the invention, SEQ ID NO. 1 is based on the mutation of the first amino acid (cysteine, C) of the fragment of amino acid residues 303 to 431 of MCF8340111.1 to alanine (A).

In the present invention, the inventors found that the first amino acid (cysteine, C) of the fragment of amino acid residues 303 to 431 of MCF8340111.1 was mutated to alanine (a) to destroy the self-cleavage activity at the N-terminus while ensuring its functionality, and thus could be used as an intein variant.

In a second aspect of the invention there is provided a nucleic acid molecule encoding a variant intein according to the first aspect of the invention.

In some embodiments of the invention, the nucleotide sequence of the nucleic acid molecule is shown in SEQ ID NO. 2.

In some embodiments of the invention, the nucleic acid molecule further comprises a sequence having more than 85% identity to SEQ ID NO. 2 and having the same function.

In some embodiments of the invention, the identity is 85%, 90%, 95%, 96%, 97%, 98% or 99%.

In a third aspect of the invention, an intein-GHK tripeptide fusion protein is provided, wherein the fusion protein comprises a sequence shown in SEQ ID NO. 1 and a GHK tripeptide sequence.

In some embodiments of the invention, the fusion protein further comprises a modification sequence.

In some embodiments of the invention, the modification sequence comprises at least one of a GST tag sequence, a Flag tag sequence, a Halo tag sequence, an HA tag sequence, a Myc tag sequence, a Snap tag sequence.

In some embodiments of the invention, the modification sequence is a His tag.

In some embodiments of the invention, the amino acid sequence of the fusion protein is shown in SEQ ID NO. 3.

In some embodiments of the invention, the nucleotide sequence of the fusion protein is shown in SEQ ID NO. 4.

In a fourth aspect of the present invention, there is provided a GHK tripeptide product comprising at least one of the following (1) to (8):

(1) Intein variants according to the first aspect of the invention;

(2) A nucleic acid molecule according to the second aspect of the invention;

(3) An expression vector comprising the nucleic acid molecule of (2);

(4) The fusion protein of the third aspect of the invention;

(5) A nucleic acid molecule encoding the fusion protein of (4);

(6) An expression vector comprising the nucleic acid molecule of (5);

(7) A transformant containing the nucleic acid molecule according to (2) or (5);

(8) A transformant containing the expression vector of (3) or (6);

in some embodiments of the invention, the transformants include viruses, bacteria, fungi, and cells.

In some embodiments of the invention, the transformant is a bacterium.

In some embodiments of the invention, the transformant is E.coli.

In some embodiments of the invention, the expression vector of (6) is derived based on the sequence set forth in SEQ ID NO. 4.

In some embodiments of the invention, the method of preparing the expression vector of (6) is: the sequence shown in SEQ ID No. 4 and the blank vector plasmid are treated by a double enzyme digestion method, and are connected by using T4 DNA ligase.

In some embodiments of the invention, the nucleotide sequence of the expression vector in (6) is shown in SEQ ID NO. 6.

In some embodiments of the invention, the transformant of (8) is prepared by: transforming bacteria with the expression vector in (6), coating the transformed bacteria on a kanamycin-containing plate, and screening positive clones.

In the invention, the inventor discovers that the intein Chi G14-GHK expression vector has a significantly higher protein expression level compared with the conventional GHK expression vector by comparing the expression difference of the intein Chi G14-GHK expression vector and the conventional GHK expression vector, so that the intein Chi G14-GHK expression vector can be used as an expression vector for high-yield GHK.

In a fifth aspect of the present invention, there is provided a method for preparing a blue copper peptide, comprising the steps of: constructing a fusion protein expression vector based on the intein and GHK tripeptide sequences shown in SEQ ID NO. 1, inducing protein expression, filtering 9-11 kDa to obtain a trapped fluid, adding copper ions for incubation, filtering 2-4 kDa, and collecting a circulating fluid to obtain the blue copper peptide.

In some embodiments of the invention, the nucleotide sequence of the fusion protein constructed based on the intein and GHK tripeptide sequences shown in SEQ ID NO. 1 is shown in SEQ ID NO. 4.

In some embodiments of the invention, the nucleotide sequence of the expression vector is shown in SEQ ID NO. 6.

In some embodiments of the invention, the agent that induces protein expression comprises IPTG (isopropyl- β -D-thiogalactoside).

In some embodiments of the invention, filtration may employ a membrane pack or a ultrafiltration tube.

In some embodiments of the invention, the first filtration is a 10KDa filter membrane filtration.

In some embodiments of the invention, the specific operation of the first filtering is: filtering with 10kDa filter membrane until the volume of the trapped fluid is less than 1/20 of the original volume, adding 20 times of the volume of the lysate into the trapped fluid, and passing through the 10kDa filter membrane again until the volume of the trapped fluid is less than 1/20 of the original volume, and repeating for multiple times.

In some embodiments of the invention, the second filtration is a 3KDa membrane filtration.

In some embodiments of the invention, the specific operation of the second filtration is: the mixture is filtered by a 3kDa filter membrane until the volume of the trapped fluid is less than 1/20 of the original volume.

In some embodiments of the invention, the copper ions are from copper salts or mixed solutions thereof with buffers.

In some embodiments of the present invention, the copper salt includes, but is not limited to, copper acetate, copper sulfate, copper chloride.

In some embodiments of the invention, the buffer includes, but is not limited to, phosphate buffer.

In some embodiments of the invention, the step further comprises concentrating and drying the flow-through.

In the present invention, the inventors found that high purity GHK-Cu can be obtained efficiently based on the above method, and the purity of GHK-Cu is as high as 98% or more. And the blue copper peptide powder obtained after drying has good appearance and stability.

In a sixth aspect of the invention there is provided the use of an intein variant according to the first aspect of the invention in the biological preparation of a blue copper peptide.

Blue copper peptide is oligopeptide composed of three amino acids, and is synthesized in a mode of adding amino acids one by one in chemical synthesis, so that the realization is complex. The inventor finds that mRNA in a 'center rule' in a natural biological system is utilized to translate into a polypeptide chain, and the polypeptide chain can orderly realize amino acid addition according to gene coding information, so that the blue copper peptide can be obtained by transferring genetic information prepared in vitro into a cell system to obtain engineering life body expression for stably expressing a genetic information product. Thus having at least the following advantages from a technical point of view over chemical synthesis: 1) The operation is simple, and the expression of the peptides can be continuously carried out only by constructing engineering bacteria for expressing the specific peptides; 2) The cost is low, the substrates are all the most basic nutrients, and expensive materials are not needed; 3) Few byproducts, easy purification, no synthesis byproducts basically, only the metabolites of living bodies, easy separation and purification; 4) Clean and environment-friendly, almost free of pollution, and meets the time requirement of carbon neutralization; 5) The efficiency is high, the synthesis efficiency is high, and the method can be used for large-scale production and acquisition. Thus, it can be seen that the related applications of the present invention have significant technical advantages over chemical synthesis. However, the problem in the prior art is that there is no case of biosynthesis of blue copper peptide because the blue copper peptide chain is too short to be detected and isolated in biological culture. Although there are methods that make it possible to use oligopeptide fusion proteins comprising protease cleavage sites to increase the length of the peptide chain of the oligopeptide, make it easy to detect the yield of the oligopeptide during expression and use affinity tags to reduce the difficulty of purification of the oligopeptide. However, this approach requires the introduction of additional endoproteases after protein purification for the release and isolation of oligopeptides. This increases the complexity of oligopeptide production and decreases the capacity of the oligopeptide. While also readily introducing additional amino acid residues, resulting in altered properties or functions of the oligopeptide. The intein of the present invention can effectively solve the above problems. Inteins are a class of amino acid sequences capable of completing cleavage by autocatalysis that are capable of completing separation between themselves and proteins under specific conditions. The intein provided by the invention can realize directional cutting aiming at GHK tripeptide, and can effectively remove various protein tags on specific proteins.

The beneficial effects of the invention are as follows:

1. the invention provides a novel intein variant sequence Chi G14, wherein a fusion protein is formed by Chi G14 and GHK, a recombinant expression vector of high-efficiency and stable blue copper peptide is prepared after a vector plasmid is introduced, and after engineering bacteria are obtained by transferring bacteria, the blue copper peptide with high purity can be obtained by induction expression.

2. The blue copper peptide is obtained based on the recombinant expression vector of the blue copper peptide, the preparation method is simple, the blue copper peptide with high purity can be efficiently obtained only through simple steps of cell disruption, ultrafiltration, intein cleavage, copper ion combination, secondary ultrafiltration and the like, the biosynthesis method can effectively reduce pollution and the generation of toxic and harmful byproducts, the yield is high, and the method is suitable for large-scale industrial mass production of the blue copper peptide and has great market value.

Drawings

FIG. 1 is a sequence alignment of Chi G14 intein with the Ssp dnaB sequence.

FIG. 2 is a predicted comparison of tertiary protein structure of Chi G14 intein and Ssp dnaB sequences, where light color is Ssp dnaB sequence and dark color is Chi G14 intein.

FIG. 3 is a plasmid map of the intein Chi G14-GHK expression vector in the examples.

FIG. 4 is a plasmid map of a conventional GHK expression vector in the examples.

FIG. 5 is an electrophoretogram of the intein expression vector induced expression of the intein G14-GHK fusion protein.

FIG. 6 is an electrophoretogram of a conventional expression vector-induced expression of a silk-protease recognition sequence-GHK fusion protein.

FIG. 7 is an electrophoretogram of the-Chi G14-GHK fusion protein obtained after Ni-NTA resin affinity chromatography purification

FIG. 8 shows the HPLC identification of the blue copper peptide product prepared using the method of the present invention.

FIG. 9 is a photograph showing the appearance of blue copper peptide powder prepared by the method of the present invention.

Detailed Description

In order to make the objects, technical solutions and technical effects of the present invention more clear, the present invention will be described in further detail with reference to the following specific embodiments. It should be understood that the detailed description is presented herein for purposes of illustration only and is not intended to limit the invention.

The experimental materials and reagents used, unless otherwise specified, are those conventionally available commercially.

Selection of Chi G14 intein design precursors

In the examples of the present invention, the inventors found that the G14 gene of Chitinophagaceae bacterium (database number: MCF 8340111.1) has a certain homology with the Ssp dnaB sequence of the conventional commercial intein (as shown in FIG. 1). Meanwhile, the inventors also predicted tertiary results for MCF8340111.1 and Ssp dnaB using AlphaFold2, which found that they were highly similar in tertiary structure, and thus hypothesized that the fragment in MCF8340111.1 may function as an intein similar to Ssp dnaB.

In this regard, the inventors cloned the fragment of the MCF8340111.1 at amino acid residues 303 to 431 as a mutation parent by a conventional method.

Construction of Chi G14 intein and application of Chi G14 intein in construction of GHK expression vector

(1) Construction of Chi G14 intein:

the first amino acid (cysteine, C) of the fragment of amino acid residues 303 to 431 of the above MCF8340111.1 was mutated into alanine (A), thereby destroying the N-terminal self-cleavage activity thereof and obtaining the intein Chi G14.

The specific amino acid sequence of the intein Chi G14 is as follows:

AVSLDTIVDIENKGLVQIKDVVVGDNILTHKGYKAISYVFPIEKQPVYRIKTKSGKEIKV SAKHKFPTLNKGLVSIDSGLTVGDELFIKNTNILLDEIESIELVGEEDTIDITVDDTHMFYAND IYTHN(SEQ ID NO:1)。

the nucleotide sequence is shown as SEQ ID NO. 2.

5’-GCAGTGTCCCTGGACACTATCGTTGATATTGAAAACAAAGGCCTGGTGCAGATC AAGGACGTAGTCGTAGGCGACAATATCCTGACTCACAAAGGCTACAAAGCGATCTCTTACGTGTTCCCGATCGAAAAACAGCCGGTTTATCGTATTAAAACTAAATCCGGTAAAGAAATCAAAGTCAGCGCTAAGCACAAATTCCCGACTCTGAACAAGGGTCTGGTTTCCATCGATAGCGGCCTGACTGTTGGTGATGAGCTGTTCATTAAAAACACTAACATCCTGCTGGATGAAATCGAATCTATCGAACTGGTTGGTGAAGAAGATACCATCGACATCACCGTAGACGACACGCACATGTTCTACGCTAACGATATCTATACCCACAAC-3’(SEQ ID NO:2)。

(2) Application of Chi G14 intein in constructing GHK expression vector:

and (3) fusing the Chi G14 intein obtained in the step (1) with a GHK tripeptide sequence, wherein the obtained fusion protein sequence is shown as SEQ ID NO. 3.

MHHHHHHAVSLDTIVDIENKGLVQIKDVVVGDNILTHKGYKAISYVFPIEKQPVYRIKT KSGKEIKV SAKHKFPTLNKGLVSIDSGLTVGDELFIKNTNILLDEIESIELVGEEDTIDITVDDT HMFYANDIYTHNGHK(SEQ ID NO:3)。

Wherein the underlined part is Chi G14 intein.

The nucleotide sequence of the fusion protein is shown as SEQ ID NO. 4.

5’-ATGCACCATCACCACCACCATGCAGTGTCCCTGGACACTATCGTTGATATTGAAA ACAAAGGCCTGGTGCAGATCAAGGACGTAGTCGTAGGCGACAATATCCTGACTCACAAAGGCTACAAAGCGATCTCTTACGTGTTCCCGATCGAAAAACAGCCGGTTTATCGTATTAAAACTAAATCCGGTAAAGAAATCAAAGTCAGCGCTAAGCACAAATTCCCGACTCTGAACAAGGGTCTGGTTTCCATCGATAGCGGCCTGACTGTTGGTGATGAGCTGTTCATTAAAAACACTAACATCCTGCTGGATGAAATCGAATCTATCGAACTGGTTGGTGAAGAAGATACCATCGACATCACCGTAGACGACACGCACATGTTCTACGCTAACGATATCTATACCCACAACGGTCACAAA-3’(SEQ ID NO:4)。

The synthesis of SEQ ID NO. 4 was submitted to Nanjing Jinsri Biotechnology Co. Of course, the person skilled in the art can choose other ways conventional in the art to synthesize according to the above sequence composition according to the actual circumstances.

Cleavage was performed at the 5 '-end of SEQ ID NO. 4 using cleavage site NcoI and at the 3' -end using cleavage site XhoI to give a cleavage fragment. Then 1. Mu.g of the blank pET28a plasmid which was also digested with NcoI and XhoI was taken (the fragment of the corresponding size was recovered using agarose gel, thus obtaining a linearized digested plasmid). The fragment and pET28a backbone were ligated overnight using T4 DNA ligase, the ligation method being performed with reference to pET28a plasmid instructions or technical manuals conventional in the art. The ligated product was transformed into BL21 (DE 3) E.coli, and the transformed E.coli was spread on a kanamycin-containing solid LB plate and cultured overnight at 37 ℃. And (3) selecting positive monoclonal in an LB liquid culture medium containing kanamycin, performing shake culture, and performing bacterial liquid PCR identification by using a vector universal primer.

Wherein, the sequence of the universal primer of the carrier is as follows: 5'-TAATACGACTCACTATAGGG-3' (SEQ ID NO: 5).

And (5) carrying out sequencing verification on the bacterial liquid after the identification, and preserving the bacterial strain with correct sequencing.

The plasmid map of the intein Chi G14-GHK expression vector is shown in FIG. 3. The nucleotide sequence of the intein Chi G14-GHK expression vector is shown in SEQ ID NO. 6.

(3) Expression differences of intein Chi G14-GHK expression vector from conventional GHK expression vector:

to highlight the expression differences of the intein Chi G14-GHK expression vector from the conventional GHK expression vector, the inventors performed a comparative comparison by inducing protein expression under the same conditions with the conventional GHK expression vector as a control.

In this embodiment, the construction method of the conventional GHK expression vector is as follows: the GHK fusion protein (SEQ ID NO: 7) comprising a silk protease recognition site was obtained by means of gene synthesis. The amino acid sequence of the GHK fusion protein comprising a silk protease recognition site is: MHHHHHHTVVGIVCRTKPDGSINVCSENDSVPVAVITKVENNN TENIEGCNSDEAVKETVGVIDSKNIVDAVHIKGSVNDVKTRTVVKQSMPDKPMAEVVKNQ PVVNDENVKGHIVGVRCPDHHHHHHPNSSSVDYGHK (SEQ ID NO: 7).

The corresponding nucleotide sequence is as follows: 5'-ATGCACCATCACCACCACCATACAGTTGTAGGAATAGTT TGTAGAACTAAACCGGACGGAAGTATCAATGTATGCTCTGAAAATGATTCAGTTCCTGTTGCTGTGATTACTAAAGTTGAAAATAATAATACAGAAAATATTGAAGGTTGTAATTCAGATGAGGCTGTAAAGGAAACAGTAGGAGTTATTGATAGCAAGAATATAGTTGATGCTGTTCACATAAAAGGCAGCGTTAATGATGTTAAAACTAGAACTGTTGTGAAGCAAAGCATGCCTGATAAGCCAATGGCTGAGGTTGTAAAGAATCAACCTGTGGTTAATGATGAAAATGTTAAGGGACATATAGTTGGGGTTAGGTGTCCGGATCATCATCACCATCACCATCCGAATTCGAGCTCCGTCGACTATGGTCACAAA-3' (SEQ ID NO: 8).

The synthesis of SEQ ID NO. 8 was submitted to Nanjing Jinsri Biotechnology Co. Cleavage was performed at the 5 '-end of SEQ ID NO. 8 using the cleavage site NcoI and at the 3' -end using the cleavage site XhoI, to obtain a cleavage fragment. Then 1. Mu.g of the blank pET28a plasmid which was also digested with NcoI and XhoI was taken (the fragment of the corresponding size was recovered using agarose gel, thus obtaining a linearized digested plasmid). The fragment and pET28a backbone were ligated overnight using T4 DNA ligase, the ligation method being performed with reference to pET28a plasmid instructions or technical manuals conventional in the art. The ligated product was transformed into BL21 (DE 3) E.coli, and the transformed E.coli was spread on a kanamycin-containing solid LB plate and cultured overnight at 37 ℃. And (3) selecting positive monoclonal in an LB liquid culture medium containing kanamycin, performing shake culture, and performing bacterial liquid PCR identification by using a vector universal primer.

Wherein the general primers for the vectors used are the same as those in the above examples. And (5) carrying out sequencing verification on the bacterial liquid after the identification, and preserving the bacterial strain with correct sequencing. The plasmid map of a conventional GHK expression vector is shown in fig. 4.

After the successfully constructed strain containing the intein Chi G14-GHK expression vector and the strain containing the conventional GHK expression vector were both cultured to OD 600=0.6 to 0.8 (0.7 in this example), IPTG (isopropyl-. Beta. -D-thiogalactoside) was added to a final concentration of 1mM, respectively, for induction expression. After centrifugation and enrichment of the expressed cells, the protein expression level was detected by SDS-PAGE electrophoresis.

The results are shown in fig. 5 and 6.

Fig. 5 shows the results of the intein expression vector induced expression of intein G14-GHK fusion protein, and fig. 6 shows the results of the conventional expression vector induced expression of silk protease recognition sequence-GHK fusion protein, and it can be found that the intein Chi G14-GHK expression vector has significantly higher protein expression level than the conventional GHK expression vector, which indicates that the intein Chi G14-GHK expression vector can be used as an expression vector for high-yield GHK.

Purification of Chi G14-GHK fusion proteins

In this example, the purification procedure for the Chi G14-GHK fusion protein based on the intein Chi G14-GHK expression vector is specifically as follows:

(1) Induction of expression:

the intein Chi G14-GHK expression vector in the above example was transformed into BL21 (DE 3) expression strain, and the monoclonal strain was selected to have an OD600 value of 0.8 in the enlarged culture in LB medium, 1/20 volume of 1M Tris-HCl buffer (pH 8.5) and IPTG at a final concentration of 1mM were added, and the culture was continued at 37℃and 200rpm for 4-6 hours. And centrifuging at 4 ℃ for 20min, and collecting thalli. The cells were collected and washed with PBS 2 times.

(2) And (3) thallus crushing:

the washed cells were resuspended in lysis buffer (consisting of final concentration of 20mM Tris and final concentration of 500mM NaCl, pH 8.0) and disrupted using pressure or ultrasound until microscopic staining confirmed that no cells were apparent. Centrifuge at 12000rpm for 20min at 4℃and collect the supernatant, and filter with 0.45 μm filter.

(3) Affinity chromatography:

after the Ni-NTA affinity chromatographic column is fully balanced by using 20 times of column volume of lysis buffer, the filtered bacterial lysate is added and transferred into the Ni-NTA affinity chromatographic column at the flow rate of 0.5mL/min, and then the Ni-NTA affinity chromatographic column is fully cleaned by using 5 times of column volume of lysis buffer containing 40mM imidazole. Eluting the target protein by using a lysis buffer solution containing 200mM imidazole with the volume of 5 times of the column volume, and obtaining the purified Chi G14-GHK fusion protein.

Purified proteins of interest were detected using SDS-PAGE, western blot and HPLC, respectively, and quantified using the BCA method.

The purification results are shown in FIG. 7.

The method can be used for efficiently purifying the Chi G14-GHK fusion protein based on Ni-NTA resin affinity chromatography, and the average capacity of the Chi G14-GHK fusion protein can reach 20mg/L through BCA quantitative detection.

Blue copper peptide GHK-Cu based on Chi G14-GHK fusion protein

In this example, the inventors obtained the blue copper peptide GHK-Cu based on the cleavage conditions and ultrafiltration of the Chi G14 intein in the above examples, and performed the following steps:

(1) Fusion protein expression:

the intein Chi G14-GHK expression vector in the above example was transformed into BL21 (DE 3) expression strain, and the monoclonal strain was selected to have an OD600 value of 0.8 in the expansion culture in LB medium, 1/20 volume of 1M Tris-HCl buffer (pH 8.5) and IPTG having a final concentration of 0.5mM were added, and the culture was continued at 37℃and 200rpm for 4-6 hours. The cells were collected by centrifugation at 4℃for 20 min. The cells were collected and washed with PBS 2 times.

(2) And (3) thallus crushing:

the washed cells were resuspended in lysis buffer (consisting of final concentration of 20mM Tris and final concentration of 500mM NaCl, pH 8.0) and disrupted using pressure or ultrasound until microscopic staining confirmed that no cells were apparent. Centrifuge at 12000rpm for 20min at 4℃and collect the supernatant, and filter with 0.45 μm filter.

(3) First round of filtration:

the filtered lysate is filtered through a 10kDa filter membrane (membrane package or ultrafiltration tube may be used) until the retentate volume is less than 1/20 of the original volume. Adding 20 times of the volume of the lysate into the trapped fluid, and passing through the 10kDa filter membrane again until the volume of the trapped fluid is less than 1/20 of the original volume. Repeated 3 times. The retentate was collected.

(4) Intein cleavage:

to the retentate, 1/3 to 1/10 volume (1/7 in this example) of a mixture of 0.5M phosphate buffer at a final concentration of pH 5.0 and 0.1 to 10mM (5 mM in this example) of copper acetate (copper sulfate or copper chloride may be used instead) was added, and after mixing, the mixture was incubated overnight at 4 ℃.

(4) Second round of filtration:

the incubated liquid is filtered through a 3kDa filter membrane (membrane package or ultrafiltration tube can be used) until the volume of the retentate is less than 1/20 of the original volume. The flow through the filter was collected. And (5) performing rotary evaporation and crystallization to obtain GHK-Cu crystals. And (5) fully drying the purified blue copper peptide to obtain blue copper peptide powder.

Taking part of the circulating liquid, and detecting the content and purity of GHK-Cu in the circulating liquid by using HPLC.

The HPLC results are shown in FIG. 8.

It was found that high purity GHK-Cu, up to 98% or more in purity, could be efficiently obtained by the method of the present example. And the blue copper peptide powder obtained after drying has good appearance and stability (figure 9).

The above examples are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above examples, and any other changes, modifications, substitutions, combinations, and simplifications that do not depart from the spirit and principle of the present invention should be made in the equivalent manner, and the embodiments are included in the protection scope of the present invention.

Claims (9)

1. An intein variant, wherein the intein variant consists ofChitinophagaceae bacteriumThe G14 gene fragment of (2) is obtained by mutation; the gene fragment is a 303-431 amino acid residue fragment of MCF 8340111.1;

the amino acid sequence of the intein variant is shown as SEQ ID NO. 1.

2. A nucleic acid molecule encoding the intein variant of claim 1.

3. The nucleic acid molecule of claim 2, wherein the nucleotide sequence of the nucleic acid molecule is set forth in SEQ ID No. 2.

4. An intein-GHK tripeptide fusion protein is characterized in that the fusion protein is a sequence shown in SEQ ID NO. 1 and a GHK tripeptide sequence.

5. The intein-GHK tripeptide fusion protein is characterized by comprising a sequence shown in SEQ ID NO. 1, a GHK tripeptide sequence and a modified sequence, wherein the modified sequence comprises at least one of a GST tag sequence, a Flag tag sequence, a Halo tag sequence, an HA tag sequence, a Myc tag sequence and a Snap tag sequence.

6. A GHK tripeptide product, comprising at least one of the following (1) - (8):

(1) The intein variant of claim 1;

(2) A nucleic acid molecule according to any one of claims 2 to 3;

(3) An expression vector comprising the nucleic acid molecule of (2);

(4) The fusion protein of claim 4 or 5;

(5) A nucleic acid molecule encoding the fusion protein of (4);

(6) An expression vector comprising the nucleic acid molecule of (5);

(7) A transformant containing the nucleic acid molecule according to (2) or (5);

(8) A transformant containing the expression vector of (3) or (6);

wherein the transformant includes viruses, bacteria and fungi.

7. A preparation method of blue copper peptide comprises the following steps: and (3) constructing a fusion protein expression vector based on the intein and GHK tripeptide sequences shown in SEQ ID NO. 1, inducing protein expression, filtering with 9-11 kDa to obtain a trapped fluid, adding copper ions for incubation, filtering with 2-4 kDa, and collecting a circulating fluid to obtain the blue copper peptide.

8. The method according to claim 7, wherein the step of concentrating and drying the flow-through solution is further performed.

9. Use of the intein variant of claim 1 for the biological preparation of a blue copper peptide.