CN113121705B

CN113121705B - Fusion protein for preparing short peptide mixture, target polypeptide, preparation method and application of short peptide mixture

Info

Publication number: CN113121705B
Application number: CN202110421051.1A
Authority: CN
Inventors: 刘霆; 刘懿; 李端忠; 刘杨昌; 杨明颜
Original assignee: Chengdu Yingpuboji Biotechnology Co ltd
Current assignee: Chengdu Yingpuboji Biotechnology Co ltd
Priority date: 2021-04-19
Filing date: 2021-04-19
Publication date: 2023-03-24
Anticipated expiration: 2041-04-19
Also published as: CN113121705A

Abstract

The invention discloses a fusion protein for preparing a short peptide mixture, a target polypeptide and a preparation method and application of the short peptide mixture; the preparation method of the short peptide mixture comprises the following steps: inserting the encoding genes of the fusion protein and the encoding genes of different proteases into corresponding vectors to obtain corresponding recombinant expression vectors, and transferring the recombinant expression vectors into corresponding host cells to obtain corresponding recombinant engineering bacteria; culturing the engineering bacteria to obtain fusion proteins and different proteases, wherein some proteases are used for enzyme digestion of the fusion proteins to obtain target polypeptides, and other proteases are used for enzyme digestion of the target polypeptides to obtain short peptide mixtures; the method has the advantages of reducing the steps and time in the subsequent separation and purification process, greatly reducing the used chemical reagents, along with low production cost, greenness, environmental protection and high efficiency.

Description

Fusion protein for preparing short peptide mixture, target polypeptide, preparation method and application of short peptide mixture

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a fusion protein for preparing a short peptide mixture, a target polypeptide, a preparation method of the short peptide mixture, and application of the fusion protein, the target polypeptide and the short peptide mixture as raw materials in preparation of cosmetics.

Background

The polypeptide is between amino acids and proteins, and is prepared by combining one or more amino acids in a certain sequence through peptide bonds. As a structural fragment of a protein, a polypeptide is involved in the biological activity of various cellular functions in an organism. With the continuous and deep understanding of the function and property of each amino acid in the polypeptide by researchers, more and more active polypeptides are fully researched and widely developed and applied to the fields of medicines, cosmetics and the like. The medicines and cosmetics mainly made of polypeptide are concerned by consumers, and the market demand is increasing.

At present, polypeptide synthesis is mainly classified into chemical synthesis methods, enzymatic methods, and recombinant DNA methods. Chemical synthesis methods are well developed and include solid phase synthesis and liquid phase synthesis, which are mainly performed by condensation between amino acids. The method has the advantages of more used organic reagents, complex steps, higher production cost, more generated impurities and difficult separation and purification. The most important disadvantage of using chemical synthesis methods is the adverse environmental impact associated with the large number of chemical reagents used in the synthesis process. The enzyme method mainly uses biological enzyme to degrade protein, and degrades animal and plant macromolecular protein into small molecular active polypeptide. Compared with a chemical synthesis method, the reaction period of polypeptide synthesis by an enzyme method is long, the yield is not high, and the requirement of industrial production is difficult to meet. The recombinant DNA method can control the sequence synthesis of the polypeptide through a proper DNA template, and the generated recombinant engineering bacteria can efficiently synthesize various active polypeptides in large quantity. Meanwhile, the method has the advantages of strong expression directionality, safe and sanitary products, wide raw material sources, low production cost and the like, and can obtain the polypeptide with high quality, safety and sanitation. In view of the huge demand for active polypeptides in commercial production, recombinant DNA methods undoubtedly satisfy a number of requirements for simple production processes, safe and hygienic products, low production costs, and the like.

GHK is an active polypeptide containing three amino acids, and its amino acid sequence is Gly-His-Lys. It is mainly present in inflammation-related proteins and extracellular matrix proteins (e.g., collagen, fibrin, etc.). There are many studies showing that GHK promotes the synthesis of extracellular matrix, elastin and structural proteins, etc., thereby promoting wound healing and tissue regeneration. Meanwhile, the composition can also enhance the differentiation and proliferation capacity of epidermal basal cells, stimulate the synthesis of decorin, promote the growth of blood vessels and nerve growth, and has certain antioxidant and anti-inflammatory functions. In addition, GHK is very easy to form GHK-Cu complex with copper ions, and the complex can effectively activate the synthesis of collagen, dermatan sulfate, chitin sulfate, small-molecule glycoprotein and glycoprotein. Therefore, GHK and GHK-Cu complex are widely applied to functional cosmetics and skin care products. GQPR is an active polypeptide consisting of four amino acids, and the amino acid sequence of the polypeptide is Gly-Gln-Pro-Arg. It is an active fragment in immunoglobulin IgG and can significantly reduce the level of inflammatory factor IL-6 in cells. It has been shown that GQPR can delay and inhibit the generation of excessive interleukin, thus inhibiting some unnecessary inflammatory reaction and glycosylation damage. In cells damaged after ultraviolet irradiation, the effect of inhibiting interleukin production is more obvious, and due to the obvious effect of inhibiting inflammatory factors, GQPR can improve lymphatic circulation, promote collagen production, prevent elastin loss, improve skin firmness and elasticity, reduce wrinkle deepening and restore skin vitality. Therefore, GQPR is widely concerned and applied in the skin care product and cosmetic industry.

As cosmetic functional ingredients well known to those skilled in the art, short peptides GHK and GQPR are often used in combination. GHK and GQPR are used as small molecular polypeptides, have small molecular weight and low immunogenicity, are not easy to cause immune reaction and have high safety. Meanwhile, the GHK and GQPR have smaller molecular volume, are easier to absorb and pass through skin, mucosa, cell membrane or other physiological barriers, and are particularly suitable for local use of human bodies. And when the GHK and the GQPR are used as active ingredients in cosmetics or skin care products in a combined way, the composition can effectively improve the aging characteristic of the skin, promote the healing of wounds and the regeneration of tissues and restore the vitality of the skin. The combination has better effect than that of single use and can generate certain gain effect. CN101277713B demonstrates that GHK, GQPR and their compositions can promote or synergistically promote the production of type I collagen by extracellular mechanisms, increase skin elasticity, prevent or improve the formation of wrinkles and sagging. Matrix of cosmetic raw material of Sadmax ^TM 3000 is a peptide mixture which is widely used in cosmetics and has the highest international market quantity, and the active ingredients of the peptide mixture are palmitoyl-glycine-glutamine-proline-arginine (Pal-GHK) and palmitoyl-glycine-histidine-lysine (Pal-GQPR), so that the peptide mixture has the efficacy of preventing or treating wrinkles. CN1893911B discloses that GHK, GQPR and derivatives thereof have a synergistic effect in promoting synthesis of type i collagen, fibronectin and hyaluronic acid, and WO2012164488A2 discloses an effect of GHK, GQPR and derivatives thereof for preventing or treating skin photoaging.

Many active polypeptides used in cosmetics are short peptides, and thus it is not generally feasible to directly produce these short peptides using recombinant methods. At present, the preparation method of GHK and GQPR is mainly a chemical synthesis method. In patent CN103665102B, a liquid phase synthesis method is provided, in which protected amino acids are synthesized one by one from N-terminal to C-segment under the action of a condensing agent, and GHK is obtained by amino acid protection, twice condensation and deprotection. However, the condensing agent is too costly, side reactions easily occur, racemization products are generated, and byproducts also easily remain in the product, which affects the quality of the final product. Traditional solid phase synthesis extends the protected amino acid from C end to N end one by one, and the final product is prepared by condensation, protection removal, recondensation, resin cutting, centrifugation, freeze-drying and high performance liquid chromatography. In patent CN105541966B, a liquid phase preparation method of GQPR is provided, which adopts multiple steps to perform condensation reaction to finally obtain the target tetrapeptide GQPR, but the preparation steps involved in the patent are up to 11 steps, which is time-consuming, costly and uses more organic reagents.

Disclosure of Invention

In order to overcome the defects, the invention provides a more green, environment-friendly and efficient preparation method of a short peptide mixture. The invention uses a recombinant DNA method to express recombinant fusion protein containing two short peptides through a strain of engineering bacteria, and a mixture of two stable short peptides GHK and GQPR with biological activity can be obtained after separation, purification and enzyme digestion. The short peptide mixture obtained by the preparation method has the activities of promoting cell proliferation and collagen generation, does not need to be further separated and purified, and can be used as a raw material to be used as an active ingredient of cosmetics and skin care products directly or added into various media. In addition, the mixture of GHK and GQPR obtained by the method can be further modified by palmitoyl to obtain Pal-GHK and Pal-GQPR for cosmetic production.

The first purpose of the invention is to provide a fusion protein for preparing a short peptide mixture, and the general structural formula of the fusion protein is thioredoxin-connecting peptide- (short peptide 1-short peptide 2) _n N is a natural number greater than or equal to 2, the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are both GHK or GQPR, and the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are different.

The second purpose of the invention is to provide a target polypeptide for preparing a short peptide mixture, which has the general structural formula of (short peptide 1-short peptide 2) _n N is a natural number greater than or equal to 2, the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are both GHK or GQPR, and the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are different.

The third objective of the present invention is to provide a method for preparing a short peptide mixture, wherein the method comprises obtaining a fusion protein for preparing the short peptide mixture by recombinant DNA technology, and obtaining a mixture of the short peptides GHK and GQPR by steps such as cleavage. Compared with a chemical synthesis method, the preparation method disclosed by the invention has the advantages of low organic reagent consumption, greenness, environmental friendliness, high preparation efficiency, short time, low production cost and the like. The preparation method comprises the following specific steps:

s1: inserting the encoding gene of the fusion protein into a vector 1 to obtain a recombinant expression vector 1, and transferring the recombinant expression vector 1 into a host cell 1 to obtain a recombinant engineering bacterium 1;

s2: inserting the coding genes of enterokinase and trypsin into the same vector 2 to obtain a recombinant expression vector 2, and transferring the expression vector 2 into a host cell 2 to obtain a recombinant engineering bacterium 2;

s3: respectively culturing to obtain

recombinant engineering bacteria

1 and 2, and separating and extracting to obtain fusion protein, enterokinase and trypsin;

s4: firstly, enterokinase is used for enzyme digestion of fusion protein to obtain target polypeptide;

s5: digesting the target polypeptide by trypsin to obtain a mixture of short peptide 1 and short peptide 2;

or comprises the following steps:

a1: inserting the encoding gene of the fusion protein into a vector 3 to obtain a recombinant expression vector 3, and transferring the recombinant expression vector 3 into a host cell 3 to obtain a recombinant engineering bacterium 3;

a2: culturing the recombinant engineering bacteria 3 to express the fusion protein, and separating and purifying to obtain the fusion protein;

a3: then, enterokinase is used for enzyme digestion of the fusion protein to obtain target polypeptide;

a4: then trypsin is used for enzyme digestion of the target polypeptide to obtain a mixture of the short peptide 1 and the short peptide 2;

or comprises the following steps:

h1: inserting the fusion protein and the coding gene of enterokinase into the same vector 4 to obtain a recombinant expression vector 4;

h2: transferring the recombinant expression vector 4 into a host cell 4 to obtain a recombinant engineering bacterium 4;

h3: culturing the recombinant engineering bacteria 4 to obtain a mixture of fusion protein and enterokinase, carrying out in-situ enzyme digestion on the fusion protein by the enterokinase, and separating and purifying to obtain target polypeptide;

h4: then trypsin is used for enzyme digestion of the target polypeptide to obtain a mixture of the short peptide 1 and the short peptide 2.

The invention has the beneficial effects that: the method has great application significance, can provide high-quality raw materials for cosmetic production, and the obtained recombinant fusion protein has excellent thermal stability, and the fusion protein can stably exist in supernatant after cell breaking by heat, while most of other protein impurities are precipitated by high-temperature heating denaturation, thereby reducing steps and time in subsequent separation and purification processes.

The fourth purpose of the invention is to provide a fusion protein, a target polypeptide and a short peptide mixture in cosmetics,

or the like, or, alternatively,

in the engineering of cell proliferation and collagen regeneration promotion,

or the like, or, alternatively,

the application in the medicines for cell proliferation and collagen regeneration promotion.

Drawings

FIG. 1 is a SDS-PAGE graph of the 15% of the expression of the fusion proteins SEQ ID Nos. 1-4 after induction;

FIG. 2 is an electrophoresis chart of a centrifugal supernatant of a recombinant engineered bacterium after heating and cell breaking (85 ℃,50 min);

FIG. 3 is an HPLC chromatogram of a reaction solution before and after enterokinase digestion of the fusion protein SEQ ID No. 2;

FIG. 4 is an HPLC chromatogram (wavelength 210 nm) of the polypeptide after ion exchange;

FIG. 5 is an HPLC chromatogram of a mixture of GHK and GQPR after trypsin digestion and membrane filtration, and a HPLC chromatogram of a commercial GHK and GQPR control (wavelength 210 nm);

FIG. 6 is an SDS-PAGE graph of the 15% of the purified recombinant enterokinase and trypsin solutions;

FIG. 7 is an HPLC chromatogram (wavelength 210 nm) of supernatant after heating and cell disruption of an induced engineering bacterium (E.coli BL21 (DE 3) PANr/pETDuet1-mTrA-L- (GHKGQPR) 3-EK);

fig. 8 is an HPLC profile (wavelength 210 nm) of a filtrate (mixture of GHK and GQPR) obtained by trypsin digestion of e.coli co-expression fusion protein SEQ ID No.1 and enterokinase polypeptide SEQ ID No. 5;

FIG. 9 is a MTT assay of NIH/3T3 cells after sample treatment;

FIG. 10 is measurement of hydroxyproline content of NIH/3T3 cells after sample treatment.

Detailed Description

Description of terms:

the terms "comprises," "comprising," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a list of steps is not limited to the steps listed, but may alternatively include steps not listed;

the terms "short peptide 1", "short peptide 2", "vector 1", "recombinant expression vector 1", "host cell 1", "recombinant engineered bacterium 1", "vector 2", "recombinant expression vector 2", "host cell 2", "recombinant engineered bacterium 2", "vector 3", "recombinant expression vector 3", "host cell 3", "recombinant engineered bacterium 3", "vector 4", "recombinant expression vector 4", "host cell 4", "recombinant engineered bacterium 4" are used for descriptive purposes only and are not to be construed as indicating or implying relative importance or implicitly indicating the number of indicated technical features.

The invention provides a fusion protein for preparing a short peptide mixture, which has a general structural formula of thioredoxin-connecting peptide- (short peptide 1-short peptide 2) _n N is a natural number greater than or equal to 2, n is preferably 3 or 4, the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are both GHK or GQPR, and the amino acid sequence of the short peptide 1 is different from the amino acid sequence of the short peptide 2; preferably, the isoelectric point of the (short peptide 1-short peptide 2) n ranges from 12 to 13 or the number of amino acids ranges from 21 to 42; the connecting peptide is any one of a protease cleavage site, a chemical substance cleavage site, a flexible peptide-protease cleavage site or a flexible peptide-chemical cleavage site; preferably, the protease cleavage sites include thrombin, 3C protease and enterokinase cleavage sites and other protease cleavage sites well known to those skilled in the art, and the chemical species include cyanogen bromide, hydroxylamine and formic acid and other protease cleavage sites well known to those skilled in the art; the cleavage site is preferably an enterokinase cleavage site;

wherein thioredoxin adopts thioredoxin (mTrA) (amino acid series shown as SEQ ID No.1 in the patent application with the publication number of CN 110257347A) which is published with the publication number of CN110257347A and is prepared according to the method disclosed in the embodiment 1 as auxiliary protein, so as to express and prepare short peptide GHK and GQPR; this thioredoxin (mTrA) has splendid thermal stability, the fusion protein who utilizes this thioredoxin (mTrA) to prepare has splendid thermal stability equally, consequently, the fusion protein is through expressing the back, only need can break the cell through one-step high temperature heating, fusion protein and other good protein of thermal stability exist in the centrifugal supernatant of the broken cell of heat, other most poor protein of thermal stability pass through high temperature heating denaturation and deposit, consequently, to obtaining the target protein, this one-step high temperature heating existing function of breaking the cell and extracting, still have the function of purification simultaneously, this has greatly promoted fusion protein extraction technology.

Preferably, the amino acid sequence of the fusion protein is shown in SEQ ID No.1 (thioredoxin mutant-enterokinase cleavage site- (GHKGQPR) _n N = 3), or as shown in SEQ ID No.2 (thioredoxin mutant-enterokinase cleavage site- (GHKGQPR) _n N = 4), or as shown in SEQ ID No.3 (thioredoxin mutant-enterokinase cleavage site- (GQPRGHK) _n N = 3), or as shown in SEQ ID No.4 (thioredoxin mutant-enterokinase cleavage site- (GQPRGHK) _n ，n＝4)；

The invention provides a target polypeptide for preparing a short peptide mixture, wherein the general structural formula of the target polypeptide is (short peptide 1-short peptide 2) n, n is a natural number which is greater than or equal to 2, n is preferably 3 or 4, the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are GHK or GQPR, and the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are different;

preferably, the amino acid sequence of the target polypeptide is shown as SEQ ID No.5 or shown as SEQ ID No.6 or shown as SEQ ID No.7 or shown as SEQ ID No. 8;

the invention provides a preparation method of a short peptide mixture, which comprises the following steps: s1: inserting the encoding gene of the fusion protein into a vector 1 to obtain a recombinant expression vector 1, and transferring the recombinant expression vector 1 into a host cell 1 to obtain a recombinant engineering bacterium 1;

s3: respectively culturing to obtain

recombinant engineering bacteria

or comprises the following steps:

h4: then, trypsin is used for enzyme digestion of the target polypeptide to obtain a mixture of the short peptide 1 and the short peptide 2.

Preferably, the host cell 1, the host cell 2, the host cell 3 and the host cell 4 in the three preparation methods are all selected from escherichia coli e.coli BL21 (DE 3) PAnr disclosed in the patent application with the publication number of CN 110396533A; coli BL21 (DE 3) PANr has resistance to multiple phages, and can fundamentally and effectively solve the problem of phage pollution.

The method can be used for preparing short peptide mixture, and directly adding into cosmetic as functional component without separation and purification for producing cosmetic.

The invention also provides the application of the fusion protein, the target polypeptide and the short peptide mixture in cosmetics or engineering or medicines for cell proliferation and collagen regeneration promotion; not limited to medical drugs and skin care essences.

The amino acid sequences of the fusion proteins and the polypeptides of interest used in the following examples 1 to 12 are shown in Table 1;

TABLE 1

Example 1

Construction of fusion protein recombinant engineering bacteria E.coli BL21 (DE 3) PANr/pET28 a-mTrA-DDK- (GHKGQPR) 3, E.coli BL21 (DE 3) PANr/pET28 a-mTrA-DDK- (GHKGQPR) 4, E.coli BL21 (DE 3) PANr/pET28 a-mTrA-DDK- (GQPRGHK) 3, E.coli BL21 (DE 3) PANr/pET28 a-mTrA-DDK- (GQPRGRGHK) 4

Step 1: artificially synthesizing amino acid sequences in the table 1, respectively inserting codon-optimized nucleotide sequences into a vector escherichia coli vector pET28a according to the amino acid sequences of fusion proteins shown in SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 to obtain 4 recombinant expression vectors;

step 2: the obtained recombinant expression vectors are respectively transformed into phage-resistant Escherichia coli E.coli BL21 (DE 3) PANr, and recombinant engineering bacteria E.coli BL21 (DE 3) PANr/pET28 a-mTrA-DDK- (GHKGQPR) 3 for expressing the fusion protein SEQ ID No.1, recombinant engineering bacteria E.coli BL21 (DE 3) PANr/pET28 a-mTrA-DDK- (GHKGQPR) 4 for expressing the fusion protein SEQ ID No.2, recombinant engineering bacteria E.coli BL21 (DE 3) PANr/pET28a-mTrA-DDDDK- (GQPRGHK) 3 for expressing the fusion protein SEQ ID No.3 and recombinant engineering bacteria E.coli BL21 (DE 3) PANr/pET28 a-mTrA-DDK- (GQPRGHK) 4 for expressing the fusion protein SEQ ID No.4 are respectively obtained.

And step 3: the cells were cultured in LB solid medium containing kanamycin (50. Mu.g/ml), and positive transformants were picked for colony PCR identification. After PCR identification, selecting a plurality of transformants with correct identification and sending out sequencing, wherein the transformants with correct sequencing are the recombinant engineering bacteria and are stored at-20 ℃ for later use.

Example 2

Expression of fusion proteins

1. Method for producing a composite material

Step 1: the engineering bacteria strains with the correct sequencing obtained in example 1 are respectively inoculated into LB liquid culture medium containing kanamycin (50 mug/ml), cultured overnight at 37 ℃ and 250rpm, the obtained fresh culture solution is used as seeds, inoculated into the fresh LB liquid culture medium containing kanamycin (50 mug/ml) in an inoculation amount of 1%, and continuously cultured at 37 ℃ and 250rpm for 6h;

step 2: adding IPTG to make the final concentration 0.4mM, and continuing culturing at 37 ℃ and 250rpm for 24h;

and step 3: centrifuging the obtained culture solution at 5000rpm for 15min, and collecting thallus;

and 4, step 4: and (3) taking 100 mu l of culture solution, centrifuging the obtained thalli, suspending the thalli in 40 mu l of deionized water, adding a certain volume of 10 mu l of 5 xSDS-PAGE sample loading buffer solution, mixing uniformly, boiling at 100 ℃ for 5min to prepare a sample, and detecting and analyzing the expression condition of the fusion protein by SDS-PAGE.

2. Results

As shown in FIG. 1, the fusion proteins SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 are expressed, specifically: the fusion proteins SEQ ID No.1 (lane 1), SEQ ID No.3 (lane 3) are expressed at about 15kD (theoretical molecular weight 14.5 kD), and the fusion proteins SEQ ID No.2 (lane 2), SEQ ID No.4 (lane 4) are expressed at about 15kD (theoretical molecular weight 15.3 kD).

Example 3

Solubility and thermostability of fusion proteins

1. Method of producing a composite material

Step 1: the E.coli cells obtained in example 2 were resuspended in 1/3V of culture medium volume of 20mM Tris-HCl (pH 7.5) and 0.2% Triton-X100, and after mixing well, heated at 85 ℃ for 50min;

step 2: centrifuging the heated suspension at 15000rpm for 8min, and collecting the centrifuged supernatant;

and step 3: the supernatant was sampled to 40. Mu.l, and 10. Mu.l of 5 XSDS-PAGE buffer was added to the supernatant, mixed well, boiled at 100 ℃ for 5min to prepare a sample, and SDS-PAGE was used to determine the solubility and thermal stability of the fusion protein.

2. Results

As shown in FIG. 2, wherein in Panel A of FIG. 2,

protein lanes

1 and 2 are centrifugal supernatants after heating and cell-breaking of the fusion proteins of SEQ ID No.1 and SEQ ID No.3, and in Panel B of FIG. 2,

protein lanes

1 and 2 are centrifugal supernatants after heating and cell-breaking of the fusion proteins of SEQ ID No.2 and SEQ ID No. 4. Compared with the total protein of the thallus before heating, after heating for 50min at 85 ℃, the fusion proteins SEQ ID No.1, SEQ ID No.2, SEQ ID No.3 and SEQ ID No.4 still exist in the supernatant stably, which indicates that the fusion proteins are expressed in Escherichia coli cells in a soluble form and have strong heat stability.

Example 4

Preparation of the polypeptide of interest

1. Method of producing a composite material

Step 1: heating and breaking the thalli obtained by the method in the embodiment 3 respectively, centrifuging by a disc centrifuge, filtering by a clarifying membrane and filtering by an ultrafiltration membrane with the cut-off molecular weight of 5kD to obtain crude extract containing fusion protein SEQ ID No.1-4 respectively;

step 2: adding reaction buffer mother liquor and enterokinase into the crude fusion protein extract to ensure that the working concentration of the reaction buffer is 100mM Tris-HCl, pH =8.5, 50mM NaCl,2mM CaCl ₂ Enterokinase concentration is 5U/mg fusion protein, and enzyme digestion reaction is carried out for 10h at 25 ℃;

and step 3: purifying the reaction solution (containing target polypeptide SEQ ID No. 5-8) after enzyme digestion by using an ion exchange chromatography, which comprises the following specific steps: the reaction solution was loaded on an equilibrium buffer well-balanced column. After the sample loading is finished, eluting with eluent 1, then washing off protein with eluent 2, finally eluting the target polypeptide with eluent 3, and collecting eluent 3. Wherein, the balance buffer:20mM sodium acetate, 50mM NaCl, pH 5.8; eluent 1:20mM Tris-HCl,100mM NaCl, pH 9.91; eluent 2:20mM Tris-HCl, 150mM NaCl, pH 9.86; eluent 3 had a composition of 20mM Tris-HCl,100mM NaCl, pH 9.86.

2. Results

The HPLC analysis in FIG. 3 shows that the fusion protein SEQ ID No.2 is only cleaved by enterokinase in the reaction solution for 0h (retention time 18.42min, FIG. 3A), whereas the fusion protein has been cleaved by enterokinase in the reaction solution to the accessory protein fragment mTrA-DDDDK (retention time 18.79min, FIG. 3B) and the polypeptide of interest SEQ ID No.6 (retention time 9.66 min, FIG. 3B) when the cleavage has proceeded for 10 h.

As shown in FIG. 4, after the centrifugation supernatants after heat cell disruption are respectively subjected to enterokinase enzyme digestion reaction and ion exchange step, the target polypeptides are respectively obtained by HPLC analysis of the eluent. The method specifically comprises the following steps: a target polypeptide SEQ ID No.5 (A picture in figure 4, retention time 9.44 min) obtained by enzyme digestion of the corresponding fusion protein SEQ ID No. 1; a target polypeptide SEQ ID No.6 obtained by enzyme digestion of the corresponding fusion protein SEQ ID No.2 (B picture in figure 4, retention time 9.48 min); a target polypeptide SEQ ID No.7 obtained by enzyme digestion of the corresponding fusion protein SEQ ID No.3 (figure 4, C picture, retention time 9.19 min); the target polypeptide SEQ ID No.8 is obtained by enzyme digestion of the corresponding fusion protein SEQ ID No.4 (FIG. 4, D, retention time 9.35 min).

Example 5

Preparation of mixture of short peptide 1 and short peptide 2

1. Method of producing a composite material

Step 1: diluting the target polypeptide obtained in the example 4 by adding water, and adjusting the pH to 8.54;

step 2: adding reaction buffer mother liquor and trypsin to ensure that the reaction buffer concentration is 50mM Tris-HCl, pH =8.2 and 50mM NaCl, and the trypsin concentration is 1U/mg target polypeptide;

and step 3: performing enzyme digestion at 25 ℃ for 10-20h;

and 4, step 4: and (3) filtering the reaction solution after enzyme digestion in the step (3) by using a 5kD ultrafiltration membrane, removing trypsin, collecting an external solution as a mixture of the short peptide 1 and the short peptide 2, storing at-20 ℃, and simultaneously sampling for HPLC analysis.

2. Results

As shown in a in fig. 5, after the target polypeptide is digested with trypsin by HPLC analysis, a mixture of short peptides GHK and GQPR is obtained, the retention time of GHK is 3.32min, the retention time of GQPR is 5.61min, and is consistent with the retention time of commercial GHK and GQPR control products (fig. 5, B and 5, C), thus proving that the mixture of GHK and GQPR is obtained by the method disclosed in example 5 and the substance obtained after the digestion with trypsin.

Example 6

Construction of recombinant engineering bacterium E.coli BL21 (DE 3) PANr/pETDuet1-EK-TRY for co-expression of enterokinase and trypsin

Step 1: artificially synthesizing amino acid sequences of enterokinase and trypsin, and respectively inserting the nucleotide sequences of the enterokinase and the trypsin which are optimized by codons into MCS1 and MCS2 of a vector pETDuet-1 to obtain a recombinant expression vector;

step 2: coli BL21 (DE 3) PANr of the recombinant expression vector transformed phage resistance, cultured in LB solid medium containing ampicillin (50. Mu.g/ml), and positive transformants were picked for colony PCR identification. After PCR identification, selecting a plurality of transformants which are identified correctly, sending out a sequence, and storing the transformants which are identified correctly, namely the recombinant engineering bacteria E.coli BL21 (DE 3) PANr/pETDuet1-EK-TRY.

Example 7

Co-expression and purification of enterokinase and trypsin

1. Method of producing a composite material

Step 1: the engineering bacteria E.coli BL21 (DE 3) PANr/pETDuet1-EK-TRY with correct sequencing obtained in example 6 is inoculated into LB liquid culture medium containing ampicillin (50 mu g/ml), cultured overnight at 37 ℃ at 250rpm, the obtained fresh culture solution is used as a seed, the inoculation amount of the seed is 1 percent, the fresh LB liquid culture medium containing ampicillin (50 mu g/ml) is inoculated, and the culture is continued for 6 hours at 37 ℃ at 250 rpm;

and 4, step 4: homogenizing the thallus by a homogenizer, denaturing the centrifugal precipitate by 8M urea, renaturing, purifying by ion exchange chromatography to respectively obtain enzyme solutions containing the recombinant enterokinase and the trypsin, and sampling and detecting and analyzing by SDS-PAGE. The two enzyme solutions are respectively stored in 50% glycerol (w/v) at-20 ℃ for later use.

And 5: the enterokinase prepared in step 4 was used to enzymatically cleave the fusion protein in example 4 to obtain a polypeptide.

Step 6: using the trypsin prepared in step 4, the desired polypeptide was cleaved by the enzyme in example 5 to obtain a mixture of short peptide 1 and short peptide 2.

The enterokinase and trypsin used in this example are commercially available products.

2. Results

As shown in FIG. 6, recombinant enterokinase and recombinant trypsin were obtained by culturing, inducing, separating and purifying the engineered bacteria, respectively. The method specifically comprises the following steps: purified recombinant enterokinase (target molecular weight 26kD, lanes 1-4 of panel A in FIG. 6) and trypsin (target molecular weight 24kD, lanes 1-4 of panel B in FIG. 6) were obtained. The obtained recombinant enterokinase is used for enzyme digestion of crude extract of fusion protein SEQ ID No.1-4, and the objective polypeptide SEQ ID No.5-8 (figure 4) is obtained by separation and purification. The obtained recombinant trypsin is used for enzyme digestion of the target polypeptide, the obtained mixture of the short peptides GHK and GQPR is shown as A diagram in figure 5, GHK retention time in figure 5: 3.32min; GQPR retention time: 5.61min; FIG. 6 is a drawing A showing recombinant enterokinase; panel B is recombinant trypsin.

Panel A in FIG. 4 is polypeptide SEQ No.5 (retention time 9.44 min); panel B shows polypeptide SEQ No.7 (retention time 9.48 min); panel C is polypeptide SEQ No.6 (retention time 9.19 min); panel D shows polypeptide SEQ No.8 (retention time 9.35 min).

Example 8

Coli BL21 (DE 3) PANr/pETDuet1-mTrA-L- (GHKGQPR) 3-EK construction for co-expression of fusion protein and enterokinase

Step 1: artificially synthesizing an amino acid sequence of fusion protein shown in a table 1, SEQ ID No.1 and an amino acid sequence of enterokinase, and jointly inserting the fusion protein subjected to codon optimization and a nucleotide sequence of enterokinase into a vector Escherichia coli vector pETDuet-1 to obtain a recombinant expression vector pETDuet1-mTrA-L- (GHKGQPR) 3-EK;

step 2: the recombinant expression vector obtained was transformed into phage-resistant E.coli BL21 (DE 3) PANr, cultured in LB solid medium containing ampicillin (50. Mu.g/ml), and positive transformants were selected for colony PCR identification. After PCR identification, selecting a plurality of transformants which are identified correctly, sending out a sequence, and storing the transformants which are identified correctly for later use, namely the recombinant engineering bacterium E.coli BL21 (DE 3) PANr/pETDuet1-mTrA-L- (GHKGQPR) 3-EK.

Example 9

Co-expression of fusion protein and enterokinase, preparation of target polypeptide and mixture of short peptide 1 and short peptide 2

1. Method of producing a composite material

Step 1: the correctly sequenced engineering bacterium E.coli BL21 (DE 3) PANr/pETDuet1-mTrA-L- (GHKGQPR) 3-EK obtained in example 8 is inoculated into LB liquid culture medium containing ampicillin (50 mu g/ml), cultured overnight at 37 ℃ and 250rpm, the obtained fresh culture solution is used as a seed, inoculated into fresh LB liquid culture medium containing ampicillin (50 mu g/ml) in an inoculation amount of 1%, and continuously cultured for 6h at 37 ℃ and 250 rpm;

step 2: adding IPTG to make the final concentration 0.4mM, and continuing culturing at 30 ℃ and 250rpm for 24h;

and 4, step 4: heating the thalli collected in the step 3 at 85 ℃ for 50min to break the thalli, centrifuging, and clarifying the supernatant to obtain a filtrate containing the polypeptide SEQ ID No. 5;

and 5: the polypeptide SEQ ID No.5 filtrate obtained in the step 4 is digested with trypsin obtained in example 7, and prepared according to the method of example 5, so as to obtain a mixture of short peptides GHK and GQPR.

2. Results

When the fusion protein SEQ ID No.1 and enterokinase are expressed in E.coli BL21 (DE 3) PANr/pETDuet1-mTrA-L- (GHKGQPR) 3-EK cells, the enterokinase enzyme-cleaves the fusion protein SEQ ID No.1 in situ to obtain the target polypeptide SEQ ID No.5. Coli BL21 (DE 3) PANr/pETDuet1-mTrA-L- (GHKGQPR) 3-EK after the culture, after the thalli are broken by heat, the centrifugal supernatant is filtered by a clarifying membrane, the HPLC analysis of the external liquid shows that the polypeptide SEQ ID No.5 (figure 7) is generated, the retention time of the polypeptide SEQ ID No.5 in figure 7: 9.64min; retention time of auxiliary protein thioredoxin-connecting peptide fragment after enterokinase in-situ enzyme digestion: 18.79min; remaining fusion protein SEQ ID No.1 retention time: 18.49min; this shows that the target polypeptide SEQ ID No.5 can be obtained by coexpression of the fusion protein and enterokinase by the engineering bacteria of Escherichia coli. After the filtrate containing the polypeptide of SEQ ID No.5 was digested with recombinant trypsin, HPLC analysis of the filtrate showed the production of short peptides GHK and GQPR (fig. 8), GHK retention time in fig. 8: 3.34min; GQPR retention time: 5.58min.

Example 10

Essence preparation for promoting collagen generation

Step 1: soaking carbomer in water for 30min, swelling, and making into 2% solution, and recording as phase A;

step 2: dissolving appropriate amount of phenoxyethanol in glycerol, adding water to obtain solution with glycerol concentration of 28% and phenoxyethanol concentration of 3%, and recording as phase B;

and step 3: mixing the phase A and the phase B according to the proportion of 1:1, and marking as phase C;

and 4, step 4: dissolving potassium sorbate in water to prepare a solution with the concentration of 0.4 percent, and marking as a phase D;

and 5: and (3) mixing the phase C and the phase D according to the proportion of 1:1, and marking as phase E;

step 6: regulating the pH value of the phase E to 6.5-7.5 by triethanolamine at the temperature of 50 ℃, and marking the obtained solution as a phase F;

and 7: adding the mixture of the short peptide GHK and the GQPR obtained in the example 5 and copper sulfate into the aqueous solution to prepare solutions with the GHK and the GQPR concentration of 20 mu m and the copper sulfate concentration of 10 mu m respectively, and fully mixing the solutions to be marked as a G phase;

and step 8: at 35 ℃, mixing the F phase and the G phase according to the ratio of 1:1 to obtain the essence containing GHK and GQPR, and storing for later use.

Example 11

Detection of cell proliferation by short peptide mixture

1. Method of producing a composite material

Step 1: on the first day, NIH/3T3 cells were dispensed into 96-well plates to a concentration of 3000 cells/well. DMEM medium with 0.4% Fetal Bovine Serum (FBS) is replaced after the adherence;

step 2: the next day, commercial GHK control, commercial GQPR control, blank medium supernatant without sample addition (negative control, NC), and mixture samples a, B, C, D of GHK and GQPR prepared according to the methods of examples 1-5 were added to DMEM medium containing 0.4% fbs (GHK and GQPR working concentrations were about 10 μ M each), mixed and added to each cell well at 100 μ L per well in 3 replicates per sample;

and step 3: adding the culture prepared in the step 2, continuing culturing at 37 ℃ for 48h, and adding 10 mu L of 5mg/mL MTT (thiazolyl blue tetrazolium bromide) into each well;

and 4, step 4: after 4 hours of incubation, the medium was carefully aspirated and discarded, and 150 μ L DMSO was added to the wells;

and 5: the culture plate is placed on a shaking table and shaken until the purple crystal is completely dissolved, and the time is about 30min to 2h;

step 6: placing the plate in a BIOTEK full-wavelength plate reader, setting detection wavelength at 570nm and reference wavelength at 630nm, measuring light absorption value, and determining relative cell activity = (detection hole: OD) ₅₇₀ -OD ₆₃₀ ) 100%/(negative control wells: OD ₅₇₀ -OD ₆₃₀ ) The relative activity of the cells was calculated.

2. Results

Sample a in fig. 9: the fusion protein SEQ ID No.1 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.5, and then is subjected to trypsin enzyme digestion and then purified to obtain a mixture of GHK and GQPR; b: the fusion protein SEQ ID No.2 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.6, and then is subjected to protease enzyme digestion and then purified to obtain a mixture of GHK and GQPR; c: the fusion protein SEQ ID No.3 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.7, and then is subjected to trypsin enzyme digestion and then purified to obtain a mixture of GHK and GQPR; d: and the fusion protein SEQ ID No.4 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.8, and then is subjected to trypsin enzyme digestion and then purified to obtain a mixture of GHK and GQPR.

As can be seen from fig. 9, the mixture of short peptides GHK and GQPR prepared according to the methods of examples 1-5 showed increased promotion of cell proliferation compared to the commercial GHK and GQPR controls. The method specifically comprises the following steps: the corresponding fusion protein SEQ ID No.1 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.5, and then a GHK and GQPR mixture obtained by trypsin enzyme digestion and purification is a sample A, and compared with NC, the cell proliferation promoting effect is 115.21%; the corresponding fusion protein SEQ ID No.2 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.6, a mixture of GHK and GQPR obtained by trypsin enzyme digestion and purification is a sample B, and compared with NC, the cell proliferation promoting effect is 126.54%; the corresponding fusion protein SEQ ID No.3 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.7, a mixture of GHK and GQPR obtained by trypsin enzyme digestion and purification is a sample C, and compared with NC, the cell proliferation promoting effect is 113.62%; and the corresponding fusion protein SEQ ID No.4 is subjected to enterokinase enzyme digestion and then purified to obtain the target polypeptide SEQ ID No.8, and a GHK and GQPR mixture obtained by trypsin enzyme digestion and purification is a sample D, and compared with NC, the cell proliferation promoting effect is 120.68%.

Example 12

Functional assay for promoting collagen production

Hydroxyproline is one of imino acids, one of the main components of collagen tissue, and is a specific amino acid in collagen, accounting for about 13% of the total amino acids in collagen. By utilizing the characteristic that the hydroxyproline has the highest content in the collagen, the hydroxyproline content measurement of blood, urine and tissues becomes an important index for measuring the collagen tissue metabolism of an organism.

1. Method of producing a composite material

Step 1: NIH/3T3 cells were dispensed into 24-well plates to a concentration of 14000 cells/well. DMEM medium containing 10% Calf Serum (CS);

step 2: the following day, sodium L-ascorbate (positive control, PC), blank medium without sample (negative control, NC), essence prepared in example 10 (sample E) and mixture of GHK and GQPR prepared according to the methods of examples 1-5, samples A, B, C and D were added to DMEM medium containing 10% CS (working concentration of GHK and GQPR was about 10. Mu.M), mixed and added to each well of cells at 1mL per well and 3 samples in parallel;

and step 3: adding the culture prepared in the step 2, continuing culturing for 72 hours at 37 ℃, and then sucking the culture medium supernatant for determining the content of hydroxyproline;

and 4, step 4: hydroxyproline content determination was performed using hydroxyproline determination kit (acid hydrolysis method) according to the procedures of the specification.

2 results

The result is shown in figure 10, compared with NC, the mixture of the short peptide GHK and GQPR obtained in example 5 can obviously stimulate the cells to generate hydroxyproline, which indicates that the mixture of the short peptide GHK and GQPR has obvious promotion effect on the mouse fibroblast NIH/3T3 collagen production. The method specifically comprises the following steps: the corresponding fusion protein SEQ ID No.1 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.5, a GHK and GQPR mixture obtained by trypsin enzyme digestion and purification is a sample A, and compared with NC, the content of hydroxyproline in cell supernatant is 137.93%; the corresponding fusion protein SEQ ID No.2 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.6, a mixture of GHK and GQPR obtained by protease enzyme digestion and purification is a sample B, and compared with NC, the content of hydroxyproline in cell supernatant is 143.68%; the corresponding fusion protein SEQ ID No.3 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.7, a mixture of GHK and GQPR obtained by trypsin enzyme digestion and purification is a sample C, and compared with NC, the content of hydroxyproline in cell supernatant is 135.63%; the corresponding fusion protein SEQ ID No.4 is subjected to enterokinase enzyme digestion and then purified to obtain the target polypeptide SEQ ID No.8, and then the mixture of GHK and GQPR which is subjected to trypsin enzyme digestion and then purified is a sample D, compared with NC, the cell proliferation promoting effect is 139.08%, and the PC is 135.63%. The mixture of the short peptides GHK and GQPR obtained by the preparation method related to the embodiments 1-5 of the invention has obvious promotion effect on the collagen production of mouse fibroblasts. The sample E (the essence containing the mixture of the short peptides GHK and GQPR prepared in the example 10) can obviously stimulate cells to secrete collagen, and compared with NC, the hydroxyproline content of the essence is as high as 152.87%, which shows that the essence has a remarkable promoting effect on the cells to secrete collagen.

Sample a in fig. 10: the fusion protein SEQ ID No.1 is subjected to enterokinase enzyme digestion and then purified to obtain a target polypeptide SEQ ID No.5, and then is subjected to trypsin enzyme digestion and then purified to obtain a mixture of GHK and GQPR; b, purifying the fusion protein SEQ ID No.2 after enzyme digestion by enterokinase to obtain a target polypeptide SEQ ID No.6, and purifying the obtained mixture of GHK and GQPR after enzyme digestion by protease; c, performing enzyme digestion on the fusion protein SEQ ID No.3 by using enterokinase, purifying to obtain a target polypeptide SEQ ID No.7, performing enzyme digestion by using trypsin, and purifying to obtain a mixture of GHK and GQPR; d, purifying the fusion protein SEQ ID No.4 after enzyme digestion by enterokinase to obtain a target polypeptide SEQ ID No.8, and purifying the obtained mixture of GHK and GQPR after enzyme digestion by trypsin; e essence prepared in example 10.

Finally, it should be noted that: the above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Sequence listing

<110> Chengdu Yingpu Boji Biotechnology Ltd

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Claims

1. A fusion protein for preparing a short peptide mixture is characterized in that the general structural formula of the fusion protein is thioredoxin-connecting peptide- (short peptide 1-short peptide 2) n, n is a natural number which is more than or equal to 2, the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are GHK or GQPR, and the amino acid sequence of the short peptide 1 and the amino acid sequence of the short peptide 2 are different;

the isoelectric point range of the (short peptide 1-short peptide 2) n is 12-13 or the number of amino acids is 21-42; the connecting peptide is any one of a protease cleavage site, a chemical substance cleavage site, a flexible peptide-protease cleavage site or a flexible peptide-chemical cleavage site;

and n is 3 or 4.

2. The fusion protein of claim 1, wherein the protease cleavage sites comprise thrombin, 3C protease, and enterokinase cleavage sites, and the chemical compound comprises cyanogen bromide, hydroxylamine, and formic acid.

3. The fusion protein of claim 2, wherein the protease cleavage site is an enterokinase cleavage site.

4. The fusion protein according to any one of claims 1-3, wherein the amino acid sequence of the fusion protein is as shown in SEQ ID No.1, or as shown in SEQ ID No.2, or as shown in SEQ ID No.3, or as shown in SEQ ID No. 4.

5. A method for preparing a short peptide mixture is characterized by comprising the following steps: s1: inserting the encoding gene of the fusion protein of claim 3 into a vector 1 to obtain a recombinant expression vector 1, and transferring the recombinant expression vector 1 into a host cell 1 to obtain a recombinant engineering bacterium 1; s2: inserting the coding genes of enterokinase and trypsin into the same vector 2 to obtain a recombinant expression vector 2, and transferring the expression vector 2 into a host cell 2 to obtain a recombinant engineering bacterium 2; s3: respectively culturing to obtain recombinant engineering bacteria 1 and 2, and separating and extracting to obtain fusion protein, enterokinase and trypsin; s4: firstly, enterokinase is used for enzyme digestion of fusion protein to obtain target polypeptide; s5: digesting the target polypeptide by trypsin to obtain a mixture of short peptide 1 and short peptide 2; or comprises the following steps: a1: inserting the encoding gene of the fusion protein of claim 3 into a vector 3 to obtain a recombinant expression vector 3, and transferring the recombinant expression vector 3 into a host cell 3 to obtain a recombinant engineering bacterium 3; a2: culturing the recombinant engineering bacteria 3 to express the fusion protein, and separating and purifying to obtain the fusion protein; a3: then, enterokinase is used for enzyme digestion of the fusion protein to obtain target polypeptide; a4: then trypsin is used for enzyme digestion of the target polypeptide to obtain a mixture of the short peptide 1 and the short peptide 2; or comprises the following steps: h1: inserting the fusion protein of claim 3 and the coding gene of enterokinase into the same vector 4 to obtain a recombinant expression vector 4; h2: transferring the recombinant expression vector 4 into a host cell 4 to obtain a recombinant engineering bacterium 4; h3: culturing the recombinant engineering bacteria 4 to obtain a mixture of fusion protein and enterokinase, carrying out in-situ enzyme digestion on the fusion protein by the enterokinase, and separating and purifying to obtain target polypeptide; h4: then trypsin is used for enzyme digestion of the target polypeptide to obtain a mixture of the short peptide 1 and the short peptide 2.