CN108948151B - Peptide and its preparation method and application - Google Patents
Peptide and its preparation method and application Download PDFInfo
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- CN108948151B CN108948151B CN201810885017.8A CN201810885017A CN108948151B CN 108948151 B CN108948151 B CN 108948151B CN 201810885017 A CN201810885017 A CN 201810885017A CN 108948151 B CN108948151 B CN 108948151B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
- A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
Abstract
The present invention relates to peptides and its preparation method and application.Peptide includes with sequence shown in SEQ ID No.1 or to replace, lack, be inserted into and/or add the sequence of one or several amino acid in sequence shown in SEQ ID No.1, and the peptide display inhibits the activity of melanoblast or melanin production or the activity of whitening.The present invention also provides the nucleotide sequence of encoded peptide, the carrier containing nucleotide sequence and include the host of carrier.Peptide of the invention is similar to the amino acid sequence of MSH- α, and can be used for skin-lightening cosmetic industry by the method large scale preparation of biofermentation.
Description
Technical field
The invention belongs to gene engineering technology fields, are related to peptide and its preparation method and application.
Background technique:
Skin-whitening is the persistent pursuit of many Asia womens, more there is traditional saying of " a white screening hundred is ugly ".From science
For angle, the color of skin is mainly since the pigment content in skin determines that main is exactly the content of melanin.Skin
The content of melanin can be largely fixed the depth of skin color in skin.Melanin is a kind of protein, is present in every
In the tissue such as individual's skin, hair, retina, pia mater.The particle of melanin is not all ater, also have yellowish-brown,
Rufous, sepia, dark-brown etc..They concur, and determine the different colours of human skin and hair.
Melanin is to be secreted by melanoblast and generated.Melanoblast belongs to gland cell, there is very strong point
Ability is secreted, the basal layer of epidermis iuntercellular of people is distributed mainly on.Melanin is got by tyrosine catalysis reaction.Melanin is female
Cell contains special tyrosinase, and oxidizing tyrosine is first formed polysaccharide, then passes through multistep metabolic process, ultimately produces black
Pigment.Melanin production it is more, skin is more black.As long as melanoblast is allowed to generate melanin less, human body skin could be allowed
Skin bleaches.Usually there are two types of thinkings to reduce the generation of melanin, and a kind of thinking is the tyrosine reduced in melanoblast
The biological activity of enzyme, second of thinking are to inhibit the activity of melanoblast by control exogenous stimulation signal.This hair
Bright whitening is exactly to take second of thinking with small peptide.
The activity of various cells generally all receives the adjusting of cell factor in human body, and melanoblast is no exception.
MSH full name is melanocyte-stimulating hormone (melanocyte-stimulating hormone) or melanotropin, is human body
In very important hormone, generated by hypophysis.The function of MSH is very powerful, can both stimulate the melanin in hair and skin
Synthesis of melanin in mother cell, and appetite and sexual stimulus can be influenced in the brain.Melanoblast surface expression one kind is black
Plain melanocortin receptor, is called MSH-R, belongs to GPCR family member.It is exactly the receptor of MSH signaling molecule.MSH hormone is from hypophysis
It after secretion, is integrated on the MSH-R receptor on melanoblast, open signal access activates the life of the melanin in downstream
At.As long as can interfere with the interaction of MSH and MSH-R, so that it may block the activation of this signal path, so that it may inhibit
The generation of dermal melanin.
Both there are no parsings to obtain the crystal structure of MSH protein binding MSH-R receptor by the mankind at present, therefore have no knowledge about
In conjunction with concrete mode.But people still can prevent MSH combination MSH- by the sequence analogues of design MSH to reach
The purpose of R.The amino acid sequence of MSH- α is SYSMEHFRWGKPV, and people devise many similar polypeptides to imitate this signal
The biological function of molecule, by preventing the MSH signaling molecule of human body itself to play activation in conjunction with MSH-R receptor.Than
Such as Nonapeptide-1, or it is called melanostatine-5, is exactly the analog of MSH- α, has been shown to have whitening
Function.Its amino acid sequence is just and the amino acid sequence of MSH- α is very much like.But in the sequence of melanostatine-5
The amino acid of 2 D types is contained in face, prepared by the method for not easily passing through biofermentation, and therefore, preparation method is also all limitation
In chemical synthesis, the cost of great number limits being widely used for this kind of product.
Therefore, there is a need in the art for the whitening peptides that can facilitate synthesis by organism.
Summary of the invention
The present invention is based partially on the following discovery of inventor: all 11 amino acid for being known as the peptide of M11 are all the days of L-type
Right amino acid, it is not only increasingly similar with the amino acid sequence of MSH- α, but also can be by the method large scale preparation of biofermentation, very
So that the skin-nourishing product of this kind of small peptide class is constantly walked close to the daily life of the common people so that ordinary people possess it is safe and stable,
Practical high-quality skin-lightening cosmetic.Inventor be also found by host cell, such as Escherichia coli with the weight of M11 peptide
Complex form expression, can effectively prevent degradation of the M11 peptide in host cell.
The present invention provides peptides, and it includes with sequence shown in SEQ ID No.1 or with sequence shown in SEQ ID No.1
The middle sequence for replacing, lack, being inserted into and/or adding one or several amino acid, peptide display inhibit melanoblast or
The activity of melanin production or the activity of whitening.
The present invention also provides the nucleotide sequences of encoded peptide.
In one embodiment, nucleotide sequence may include with sequence shown in SEQ ID No.3.
The present invention also provides the carriers comprising nucleotide sequence.
The present invention also provides the host cells comprising carrier.Preferably, it is thin to can be escherichia coli host for host cell
Born of the same parents.
The present invention also provides the methods for preparing peptide comprising:
(1) host cell, preferably e. coli host cell are converted with the carrier comprising polynucleotide sequence, wherein described
Polynucleotide sequence coding polypeptide, the polypeptide include M11 peptide；
(2) host cell is cultivated under suitable condition；And
(3) collect and purify the polypeptide of expression；
(4) optionally cutting polypeptide to obtain M11 peptide of the invention, wherein it is described cutting preferably with hydroxylamine cleavage method into
Row.
In one embodiment, polynucleotide sequence coding polypeptide, the polypeptide include 1 with SEQ ID No.1 institute
The sequence shown or to replace, lack, be inserted into and/or be added to one or several amino acid in sequence shown in SEQ ID No.1
Sequence or it is multiple with sequence shown in SEQ ID No.1 or in sequence shown in SEQ ID No.1 replace, lack, insertion
And/or it is added to the repetitive unit of the sequence of one or several amino acid.In one embodiment, polynucleotide sequence coding
2,3 or 4 repetitive units with sequence shown in SEQ ID No.1.Repetitive unit can be continuously or can be spaced one
A or more amino acid.
The present invention provides skin-whitening compositions, and it includes according to peptide.
The present invention provides peptides for inhibiting the activity of melanoblast or the purposes of melanin production.
The present invention provides the purposes that peptide is used for whitening.
Compared with prior art, the invention has the characteristics that:
1, peptide disclosed in this invention does not contain any unnatural amino acid, more like with human body native protein MSH- α, is
The sequence of long-term screening and optimizing, and good water solubility, stability are strong.
2, the preparation method of novel whitening small peptide disclosed in this invention is suitable for big rule using escherichia expression system
Mould amplification, production cost is very low, and can be further improved yield by the expressing in series of polypeptide gene.Tradition is more relatively
The chemical synthesis process of peptide has significant cost advantage.
3, the preparation method of novel whitening small peptide disclosed in this invention, product purity is high, utilizes mass spectrometry method detection point
Son amount is correct, without obvious impurity component.
Novel whitening small peptide M11 good water solubility disclosed by the invention, stability is strong, purity is high, simple production process, cost
It is cheap, skin whitening product can be allowed to walk close to the daily life of the common people, have a vast market application prospect.
Detailed description of the invention
Fig. 1 is the plasmid map schematic diagram of expression vector of the present invention, and the expression vector of specific choice is pET-32a, selection
Tandem sequence repeats number be 2, i.e. pET-32a-M11-2 expression vector.
Fig. 2 is the hydroxylamine cleavage effect that the present invention gives expression to fusion protein, and the expression vector of specific choice is pET-32a-
M11-2.Peptide concentration is about 1mg/ml after digestion.
Fig. 3 is the Mass Spectrometer Method result for the M11 polypeptide that the present invention is purified into.The theoretical molecular weight of M11 polypeptide is
1378.61Da.With Matrix Assisted Laser Desorption lonization-Time of Flight instrument MALDI-TOF AXIMA-CFR Plus
(KRATOS Analytical, Shimadzu corporation, Japan) is analyzed under linear model, display polypeptide point
Son amount is correct, without obvious impurity, does not degrade.
Fig. 4 shows the comparative situation of melanin content after smearing whitening small peptide M11 or pure water.Ordinate isThe melanin content that 18 skin pigment instrument of MX measures.
Specific embodiment
It has been described in more detail below the present invention.
Peptide of the invention includes basic unit M11, is made of 11 L-type amino acid.Amino acid sequence are as follows: Gly-Met-
Pro-Phe-Arg-Trp-Phe-Lys-Pro-Val-Asn, or it is abbreviated as GMPFRWFKPVN, herein also with SEQ ID
No.1 is indicated.The theoretical molecular weight of M11 is 1378.61Da.
It can be by the way that polypeptide gene expressing in series to be significantly improved to polypeptide expression efficiency.Concatenated number can be from integer
Optimum choice in n is expressed as M11-n, the integer that wherein n is 1 or more, such as 2,3,4,5 or 6.Herein, 1 it is duplicate
Gene representation is M11-1, and amino acid sequence is GMPFRWFKPVN (SEQ ID No.1)；2 duplicate gene representations are M11-
2, amino acid sequence is GMPFRWFKPVNGMPFRWFKPVN (SEQ ID No.2)；3 duplicate gene representations are M11-3, ammonia
Base acid sequence is GMPFRWFKPVNGMPFRWFKPVNGMPFRWFKPVN (SEQ ID No.4)；4 duplicate gene representations are
M11-4；Amino acid sequence is GMPFRWFKPVNGMPFRWFKPVNGMPFRWFKPVNGMPFRWFKPVN (SEQ ID No.5)；
And so on.Therefore, peptide of the invention can also be expressed as [GMPFRWFKPVN]n, wherein n be 1 or more integer.
It can carry out preparing peptide of the invention by a variety of production technologies.Can by traditional chemically synthesized technique into
Row preparation, can also carry out recombinant expression preparation by the technique of fermentation, such as utilize Escherichia coli fermentation.
Peptide of the invention can be by nucleotide sequence coded, such as by SEQ ID No.3 (ggcatgccgtttcgctggt
Tcaaaccggtgaac) nucleotide sequence coded.It should be appreciated that those skilled in the art are easy due to Codon degeneracy
Change nucleotide sequence under the premise of retaining initial amino acid sequence.Nucleotide sequence can be cloned by routine techniques
In expression vector, then expression vector is transformed into host cell.Method for transformation includes but is not limited to electroporation, CaCl2Turn
Change etc..In the present invention, expression vector can be pET26, the common carriers such as pET32, pGEX-6p.
Peptide of the invention can include but is not limited to that zooblast, plant are thin by different organism expressings, the organism
Born of the same parents, microbial cell, such as prokaryotes and eucaryote.Preferably, in the present invention it is generated using microbial cell fermentation
Peptide of the invention.Microbial cell can be Enterobacteriaca cells, such as Bacillus coli cells, such as BL21.It should be appreciated that this
Field technical staff can choose cell appropriate to express peptide of the invention.
It can be prepared by a conventional method and generate peptide of the invention.For example, can be in the medium in condition appropriate
Lower culture conversion has the host cell of polynucleotides of the invention, such as Escherichia coli；Then pass through conventional separation and purifying skill
Art separates peptide of the invention.Separation and purification technique include but is not limited to dialysis, ammonium sulfate precipitation, high performance liquid chroma- tography.
Peptide of the invention include with sequence shown in SEQ ID No.1 or in sequence shown in SEQ ID No.1 replace,
The sequence of one or several amino acid is lacked, is inserted into and/or adds, the peptide display inhibits melanoblast or melanin raw
At activity or whitening activity." several " can be 2,3,4,5,6,7,8,9,10 or 11.
Amino acid addition refers in amino acid sequence, for example, SEQ ID NO:1 sequence inside addition or in amino acid
The C-terminal or N-terminal of sequence add amino acid, as long as peptide display inhibits the activity or beauty of melanoblast or melanin production
White activity.
Amino acid substitution refers in amino acid sequence, for example, SEQ ID NO:1 sequence some position some amino acid
Residue is substituted by other amino acid residues, as long as peptide display inhibits the activity or beauty of melanoblast or melanin production
White activity.
Amino acid insertion refers to residual in any position of the amino acid sequence such as sequence of SEQ ID NO:1 insertion amino acid
Base, the amino acid residue of insertion can also be completely or partially adjacent to each other, or between the amino acid of insertion it is not adjacent to each other, only
The peptide display is wanted to inhibit the activity of melanoblast or melanin production or the activity of whitening.
Amino acid deletions refer to can delete 1,2 or 3 or more from amino acid sequence, such as the sequence of SEQ ID NO:1
Amino acid, as long as peptide display inhibits the activity of melanoblast or melanin production or the activity of whitening.
It is known to those skilled in the art that peptide of the present invention can one or more positions between amino acid sequence
Carry out posttranslational modification.The example of posttranslational modification may include phosphorylation, acetylation and deamidization.
The present invention also provides the analogs of peptide shown in SEQ ID NO:1, as long as analog display inhibits melanin female thin
The activity of the activity or whitening of born of the same parents or melanin production.These analogs and natural peptide difference can be on amino acid sequence
Difference is also possible to not influence the difference on the modified forms of sequence, or haves both at the same time.These peptides include natural or induction
Genetic variant.Induction variant can be obtained by various technologies, such as be generated at random by radiating or being exposed to mutagens
Mutagenesis can also pass through the technology of site-directed mutagenesis or other known molecular biology.Analog further includes having to be different from naturally
The analog of the residue (such as D- amino acid) of l-amino acid, and with non-naturally occurring or synthesis amino acid (such as β, γ-
Amino acid) analog.
In the present invention, replace and can be conserved amino acid substitution, refer to compared with the amino acid sequence of SEQ ID NO:1,
There are 3, more preferably 2 amino acid or 1 amino acid are replaced by amino acid with similar or analogous properties and forms peptide.These are protected
Keeping property variation peptide can carry out amino acid substitution according to table 1 and generate.
| Initial residue | Represent the substitution of part | It is preferred to replace |
| Ala(A) | Val；Leu；Ile | Val |
| Arg(R) | Lys；Gln；Asn | Lys |
| Asn(N) | Gln；His；Lys；Arg | Gln |
| Asp(D) | Glu | Glu |
| Cys(C) | Ser | Ser |
| Gln(Q) | Asn | Asn |
| Glu(E) | Asp | Asp |
| Gly(G) | Pro；Ala | Ala |
| His(H) | Asn；Gln；Lys；Arg | Arg |
| Ile(I) | Leu；Val；Met；Ala；Phe | Leu |
| Leu(L) | Ile；Val:Met；Ala；Phe | Ile |
| Lys(K) | Arg；Gln；Asn | Arg |
| Met(M) | Leu:Phe:Ile | Leu |
| Phe(F) | Leu；Val:Ile；Ala；Tyr | Leu |
| Pro(P) | Ala | Ala |
| Ser(S) | Thr | Thr |
| Thr(T) | Ser | Ser |
| Trp(W) | Tyr；Phe | Tyr |
| Tyr(Y) | Trp；Phe；Thr:Ser | Phe |
| Val(V) | Ile；Leu；Met；Phe；Ala | Leu |
The whitening active of peptide of the invention inhibits the activity of melanoblast or melanin production that can refer to " Cui Huan
The effect of lotus, the whitening class cosmetics such as Cao Rui, Yin Jiazhen, evaluates [J] fragrance flavor and cosmetic, 2012,4 (2): 37-40 " (its
It is incorporated herein by reference) it carries out.By18 skin pigment instrument of MX is (public by German Courage+Khazaka
The production of (CK) company, department) measure melanin content.In short, test article is used continuously in subject, respectively use 1 time sooner or later daily,
Application after face cleaning.Other skin-lightening cosmetics are deactivated during the experiment.Inquire volunteer using feeling after given the test agent simultaneously
And whether there is or not allergic phenomena etc., itself front and back comparison is recorded.Determination of the environment: test environment temperature (about 22 DEG C, humidity about 50%, and
And carry out real-time dynamic monitoring.It uses18 skin pigment instrument of MX measures skin according to the directions for use of manufacturer
Skin dark red pigment content follow-on test 3 times, is averaged.
The present invention also provides a kind of nucleic acid molecules, the nucleic acid molecules include the nucleic acid sequence of coding peptide of the invention.
Nucleic acid can be DNA or cDNA.Nucleic acid molecules can be mainly made of the nucleic acid sequence for encoding peptide of the present invention, or can be only
It is formed by encoding peptide of the present invention.Such nucleic acid molecules can be synthesized with methods known in the art.Due to genetic code
Degeneracy, it will be understood by those skilled in the art that the nucleic acid molecules of different nucleic acid sequence can encode identical amino acid sequence.
It include nucleic acid sequence of the present invention in the carrier the present invention also provides carrier.Suitable carrier is to carry
Known to body building field, selection and other controlling elements including promoter, such as enhancer element.Load of the present invention
Body includes the sequence for being adapted for introduction into cell.For example, carrier can be expression vector, and in the carrier, the code sequence of the polypeptide
The control by own cis-acting regulatory element is arranged, the gene integration or gene replacement of host cell are convenient in the design of carrier
Deng.
It will be recognized by one of ordinary skill in the art that in the present invention, term " carrier " includes DNA molecular, for example, plasmid,
Bacteriophage, virus or other carriers, it contains one or more heterologous or recombination nucleic acid sequences.Suitable bacteriophage and disease
Poisonous carrier includes, but are not limited to: lambda phage, EMBL bacteriophage, simian virus, cattle wart virus, Epstein-Barr virus, gland
Virus, herpesviral, mouse sarcoma virus, murine mammary tumor virus, slow virus etc..
The production method of peptide
The production method of peptide of the invention may include steps of:
(1) genetic engineering bacterium, such as the building of Recombinant organism；
(2) fermented and cultured and inducing expression of genetic engineering bacterium；
(3) purifying and digestion of polypeptide；
(4) optionally cutting polypeptide is to obtain peptide of the invention.
By taking Recombinant organism as an example, the construction step in step (1) is as follows: optimum choice peptide M11-n's
Segment is carried out codon optimization using the method for PCR and splicing recombinates, obtains complete recombinant DNA sequence by DNA fragmentation；It will
Recombinant DNA sequence is transferred in E. coli expression strains, and screening obtains Recombinant organism.
The fermented and cultured and expression induction step of the Recombinant organism described in step (2) are as follows:
(1) the Recombinant organism single colonie from picking in LB plate after preferred, is placed in the LB culture medium of 10ml
In, 220rpm, 37 DEG C of culture 12h-16h；
(2) bacterium solution is inoculated into amplification in LB culture medium according to the ratio of 1:100 to cultivate, 220rmp, 37 DEG C of cultures 3 are small
When, when the OD600 of bacterium solution is about 0.6, the IPTG that final concentration of 0.5mM is added is induced, and 16 DEG C are continued culture 20 hours,
Thalline were collected by centrifugation.
In step (3), the purification step of peptide is as follows:
(1) bacterium is resuspended with Tris buffer, supernatant is collected by centrifugation in ultrasonication；
(2) it is purified from supernatant using affinity column and obtains the fusion protein of recombination M11-n；
(3) hydroxylamine cleavage method crack fusion protein is used, free M11 peptide is released；
(4) pass through the M11 peptide in the methods of dialysis, reversed-phase column purification solution.
Peptide of the invention may be used as whitening peptide.Such as after degerming being filtered with 0.22 μm of filter membrane, use purified water
It is diluted to the solution of 40 μ g/mL, it is for a person to use.After carrying out normal individual trial, practical application effect is good, and whitening effect is significant, safety
It is without side-effects, it can be used for a long time.
Embodiment
Following embodiment is provided further to illustrate the present invention, but it should be understood by those skilled in the art that these embodiments
It is not intended to limit the scope of the invention, is only limited by the claims that follow.
The building and expression of embodiment 1:pET-32a-M11-2 expression vector
(1) building of Recombinant organism
1.1. can be since by the way that M11 polypeptide gene expressing in series is significantly improved polypeptide expression efficiency, this embodiment be selected
N=2, that is, 2 duplicate M11-2 are selected, amino acid sequence is GMPFRWFKPVNGMPFRWFKPVN (SEQ ID No.2).
1.2. according to the amino acid sequence of M11-2, the codon gene of its Escherichia coli preference of optimum choice, that is, g
gcatgccgtttcgctggttcaaaccggtgaacggcatgccgtttcgctggtttaaaccggtgaac(SEQ ID
No.6), in order to which AAT (azanol restriction enzyme site) is added in the demand of subsequent polypeptides digestion, 5 ' ends of this gene, TAA is added in 3 ' ends
(terminator codon), complete gene areaatggcatgccgtttcgctggttcaaaccggtgaacggcatgccgtttcgc
tggtttaaaccggtgaactaa(SEQ ID No.7) (synthesis of GENEWIZ company).
Gene M 11-2 is passed through BamH I (NEB company article No.: R0136L) and Xho I by 1.3 commission GENEWIZ companies
The restriction enzyme site of (NEB company, article No.: R0146L) is inserted into PET32a expression vector (GENEWIZ company), and building obtains pET-
32a-M11-2 expression vector.
Recombinant expression carrier pET-32a-M11-2 is transformed into E. coli expression strains BL21 (Merck company) by 1.4,
Screening obtains Recombinant organism.Detailed process are as follows: 1: take the plasmid of 1 μ l in the E. coli competent of 100 μ l
In cell BL21 (DE3), 30min is stood on ice.2: by the mixture in 42 DEG C of water-baths heat shock 90s, be then immediately placed in
2min is stood on ice.3: the LB culture medium of 600 μ l non-resistants being added into the mixture, 37 DEG C, 1h is cultivated under the conditions of 220rpm.
4: the 200 μ l bacterium solutions being taken uniformly to be coated on the LAB plate containing amicillin resistance (10g/L peptone, 5g/L yeast
Extract, 10g/L sodium chloride, 15g/L agar, 100 μ g/ml ammonia benzyl antibiotic).5: plate inversion is incubated at 37 DEG C of incubators
In, culture about 20h waits growing high-visible bacterium colony.
(2) fermented and cultured and inducing expression of Recombinant organism
2.1. the Recombinant organism M11-2 single colonie from picking in LB plate after preferred, is placed in the LB of 10ml
In culture medium (10g/L peptone, 5g/L yeast extract, 10g/L sodium chloride), 220rpm, 37 DEG C of culture 12h-16h.
2.2. bacterium solution is inoculated into amplification in LB culture medium according to the ratio of 1:100 to cultivate, 220rmp, 37 DEG C of cultures 3 are small
When, when the OD600 of bacterium solution is about 0.6, the IPTG that final concentration of 0.5mM is added is induced, and 16 DEG C are continued culture 20 hours,
Thalline were collected by centrifugation.
(3) purifying and digestion of recombinant polypeptide
3.1 are resuspended bacterium (the dry bacterium/100ml of 5g) with Tris buffer (25mM Tris, 150mM NaCl, pH 7.5), surpass
Sound is broken, and (ultrasonic 4s stops 8s, stops 10min after power 100W, ultrasonic 10min, and repeat 1~2 time, until bacterium solution is limpid not
It is sticky), supernatant is collected in centrifugation (4 DEG C, 14500g is centrifuged 30 minutes), and the albumen expressed at this time is HIS6- Trx-M11-2's melts
Hop protein.
3.2 utilize nickel ion affinity column (Qiagen company, article No.: 30210.25mM Tris, 150mM NaCl are used in advance,
7.5 solution equilibria of pH) fast separating and purifying obtains HIS from supernatant6The fusion protein of-Trx-M11-2.Pass through 20mM miaow
The Tris buffer (20mM imidazoles, 25mM Tris, 150mM NaCl, pH 7.5) of azoles cleans about 10~20 cylinders of foreign protein
Product, with Tris buffer (250mM imidazoles, 25mM Tris, 150mM NaCl, pH 7.5) cleaning purpose egg of 250mM imidazoles
White, about eluting 5 column volumes can elute destination protein completely.
3.3 use hydroxylamine cleavage method crack fusion protein, release free M11 small peptide.In short, by HIS6-Trx-
The fusion protein solution and hydroxylamine cleavage liquid (4M azanol, PH9.0) of M11-2 is mixed according to the volume ratio of 1:1, is adjusted and is arrived PH
9.0, concussion reaction is closed under 45 degree of water baths, about 4~6h can be with fully reacting.Hydroxylamine solution can be in weakly alkaline environment
The site Asn-Gly for cutting active site of protein open of lower specificity, just can be by HIS6In the fusion protein of-Trx-M11-2
M11 polypeptide releases.Pass through SDS-PAGE electrophoresis detection endonuclease reaction progress.Detailed process are as follows: 40 μ l of protein liquid is taken,
Be added 10 μ l 5 × albumen sample-loading buffer (Tris-HCl (pH:6.8) of 250mM, 10%SDS, 0.5% bromophenol blue, 50%
Glycerol, 5% beta -mercaptoethanol), it is placed in 100 DEG C of boiling water and boils 10min, then every 10 μ l of hole is added in SDS-PAGE protein adhesive,
After voltage 80V runs 2h, with coomassie brilliant blue staining liquid (0.1% coomassie brilliant blue R_250,25% isopropanol, 10% glacial acetic acid)
Protein staining 20min is carried out, protein decolouring liquid (10% acetic acid, 5% ethyl alcohol) is recycled to decolourize.
The 3.4 polypeptide M11 released can further be purified by the methods of dialysis, then carry out Mass Spectrometer Method
Or freeze-drying is spare.Detailed process are as follows: polypeptide M11 is transferred in the dialysis band of 1kDa molecular cut off, is placed into 100 times thoroughly
In the distilled water solution for analysing liquid product, dialyse for 24 hours in 4 degree of environment, it is lasting to stir, primary distilled water can be changed every 4h.So
The solution in bag filter is collected afterwards, takes supernatant, M11 polypeptide as after purification after centrifugation.
The theoretical molecular weight of 3.5M11 polypeptide is 1378.61Da, can not be detected, be needed by conventional SDS-PAGE
It is identified using mass spectrometry method.As described in Figure 3, it is identified according to mass spectrometry method, the actual molecular weight of the polypeptide of M11 of the invention
It is consistent with theoretical molecular weight for 1378Da.
Embodiment 2: the normal individual trial of whitening small peptide M11
By 0.22 μm of membrane filtration degerming of whitening small peptide M11,40 μ g/mL are diluted to pure water, storage refrigerator is stand-by.
40 volunteers (male 20, women 20, the age is 23-45 years old) are randomly divided into two groups of A, B, 20 each (male
Property 10, women 10), the whitening small peptide M11 of face 40 μ g/mL of 7ml on probation on the left of A group volunteer, B group volunteer left side
Face 7ml pure water on probation, right side face is as blank control group.Other skin-lightening cosmetics are deactivated during the experiment, are continuously made
Melanin content is measured with after 7 days.
Method reference: " the effect of whitening class cosmetics such as Cui Huanlian, Cao Rui, Yin Jiazhen, evaluates the makeup of [J] spices and essence
Product, 2012,4 (2): 37-40 " (it is incorporated herein by reference), by18 skin pigment instrument of MX is (by Germany
The production of (CK) company, Courage+Khazaka company) measure melanin content.In short, test article is used continuously in subject, often
It is respectively used 1 time sooner or later, application after face cleaning.Other skin-lightening cosmetics are deactivated during the experiment.Simultaneously inquire volunteer's use by
It is felt after test agent and whether there is or not allergic phenomena etc., records itself front and back comparison.Determination of the environment: test environment temperature (about 22
DEG C, humidity about 50%, and carry out real-time dynamic monitoring.It uses18 skin pigment instrument of MX is according to manufacturer
Directions for use measure skin dark red pigment content, follow-on test 3 times, be averaged.Numerical value in Fig. 4 is 20 subjects'
Average value.
Fig. 4 shows smear whitening small peptide M11 or pure water after melanin content comparative situation, it was demonstrated that smearing whitening
Melanin content is obviously than smearing the low of pure water and blank control group after small peptide M11.
In addition, A group volunteer can obviously observe that left side face is pale compared with right side face；B group volunteer's left and right sides
Without significant change, volunteer indicates to feel good and without allergic phenomena in portion.It is concluded that M11 whitening effect is significant, nothing
Side effect.
Sequence table
<110>Shanxi Jin Bo Biomedics Inc.
<120>peptide and its preparation method and application
<130> C18P2078
<160> 7
<170> PatentIn version 3.5
<210> 1
<211> 11
<212> PRT
<213>artificial sequence
<220>
<223> M11
<400> 1
Gly Met Pro Phe Arg Trp Phe Lys Pro Val Asn
1 5 10
<210> 2
<211> 22
<212> PRT
<213>artificial sequence
<220>
<223> M11-2
<400> 2
Gly Met Pro Phe Arg Trp Phe Lys Pro Val Asn Gly Met Pro Phe Arg
1 5 10 15
Trp Phe Lys Pro Val Asn
20
<210> 3
<211> 33
<212> DNA
<213>artificial sequence
<220>
<223> M12 DNA
<400> 3
ggcatgccgt ttcgctggtt caaaccggtg aac 33
<210> 4
<211> 33
<212> PRT
<213>artificial sequence
<220>
<223> M11-3
<400> 4
Gly Met Pro Phe Arg Trp Phe Lys Pro Val Asn Gly Met Pro Phe Arg
1 5 10 15
Trp Phe Lys Pro Val Asn Gly Met Pro Phe Arg Trp Phe Lys Pro Val
20 25 30
Asn
<210> 5
<211> 44
<212> PRT
<213>artificial sequence
<220>
<223> M11-4
<400> 5
Gly Met Pro Phe Arg Trp Phe Lys Pro Val Asn Gly Met Pro Phe Arg
1 5 10 15
Trp Phe Lys Pro Val Asn Gly Met Pro Phe Arg Trp Phe Lys Pro Val
20 25 30
Asn Gly Met Pro Phe Arg Trp Phe Lys Pro Val Asn
35 40
<210> 6
<211> 66
<212> DNA
<213>artificial sequence
<220>
<223> M11-2 DNA
<400> 6
ggcatgccgt ttcgctggtt caaaccggtg aacggcatgc cgtttcgctg gtttaaaccg 60
gtgaac 66
<210> 7
<211> 72
<212> DNA
<213>artificial sequence
<220>
<223>the M11-2 coded sequence of AAT and TAA is added
<400> 7
aatggcatgc cgtttcgctg gttcaaaccg gtgaacggca tgccgtttcg ctggtttaaa 60
ccggtgaact aa 72
Claims (12)
1. by the peptide formed with sequence shown in SEQ ID No. 1.
2. encoding the nucleotide sequence of peptide according to claim 1.
3. nucleotide sequence according to claim 2, by being formed with sequence shown in SEQ ID No. 3.
4. including the carrier of nucleotide sequence according to claim 2 or 3.
5. including the host cell of carrier according to claim 4.
6. host cell according to claim 5 is e. coli host cell.
7. the method for preparing peptide according to claim 1 comprising:
(1) host cell is converted with the carrier comprising polynucleotide sequence, wherein the polynucleotide sequence coding polypeptide, institute
Stating polypeptide includes peptide described in claim 1；
(2) host cell is cultivated under suitable condition；And
(3) collect and purify the polypeptide of expression；
(4) cut polypeptide to obtain peptide according to claim 1,
Wherein the polypeptide includes multiple repetitive units with sequence shown in SEQ ID No. 1；
Plurality of repetitive unit is 2,3 or 4 repetitive units.
8. according to the method described in claim 7, wherein the host cell is e. coli host cell.
9. according to the method described in claim 7, wherein the cutting is carried out with hydroxylamine cleavage method.
10. method for claim 7, wherein the repetitive unit is continuous or the one or more amino acid residues in interval.
11. skin-whitening composition, it includes peptides according to claim 1.
12. the purposes that peptide described in claim 1 is used for whitening.
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| CN112300249B (en) * | 2020-10-30 | 2023-03-28 | 润辉生物技术（威海）有限公司 | Hair-blacking and hair-growing peptide and preparation method and application thereof |
| CN115806586B (en) * | 2022-06-15 | 2023-10-20 | 山西锦波生物医药股份有限公司 | Whitening short peptide and preparation method thereof |
Citations (5)
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| WO1994004674A1 (en) * | 1992-08-21 | 1994-03-03 | Jarl Wikberg | Human melanocyte stimulating hormone receptor |
| CN101076348A (en) * | 2004-10-08 | 2007-11-21 | 克里纽沃药物有限公司 | Compositions and methods for inducing melanogenesis in a subject |
| WO2008025074A1 (en) * | 2006-08-28 | 2008-03-06 | Clinuvel Pharmaceuticals Limited | Method for reducing incidence or rate of development of skin cancers and related conditions |
| WO2009003034A1 (en) * | 2007-06-27 | 2008-12-31 | The Board Of Trustees Of The Leland Stanford Junior University | Oligopeptide tyrosinase inhibitors and uses thereof |
| CN106999399A (en) * | 2014-09-01 | 2017-08-01 | 法国居里学院 | Skin brightening peptide agent |
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2018
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| WO1994004674A1 (en) * | 1992-08-21 | 1994-03-03 | Jarl Wikberg | Human melanocyte stimulating hormone receptor |
| CN101076348A (en) * | 2004-10-08 | 2007-11-21 | 克里纽沃药物有限公司 | Compositions and methods for inducing melanogenesis in a subject |
| WO2008025074A1 (en) * | 2006-08-28 | 2008-03-06 | Clinuvel Pharmaceuticals Limited | Method for reducing incidence or rate of development of skin cancers and related conditions |
| WO2009003034A1 (en) * | 2007-06-27 | 2008-12-31 | The Board Of Trustees Of The Leland Stanford Junior University | Oligopeptide tyrosinase inhibitors and uses thereof |
| CN101795702A (en) * | 2007-06-27 | 2010-08-04 | 里兰斯坦福初级大学理事会 | Oligopeptide tyrosinase inhibitors and uses thereof |
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| Melanocortin Receptor Ligands: New Horizons for Skin Biology and Clinical Dermatology;Bohm M.等;《Journal of Investigative Dermatology 》;20061231;全文 |
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| CN108948151A (en) | 2018-12-07 |
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