CN109608518B - Pentapeptide and application thereof - Google Patents
Pentapeptide and application thereof Download PDFInfo
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- CN109608518B CN109608518B CN201811311659.3A CN201811311659A CN109608518B CN 109608518 B CN109608518 B CN 109608518B CN 201811311659 A CN201811311659 A CN 201811311659A CN 109608518 B CN109608518 B CN 109608518B
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- serum albumin
- pentapeptide
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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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- B—PERFORMING OPERATIONS; TRANSPORTING
- B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
- B01J—CHEMICAL OR PHYSICAL PROCESSES, e.g. CATALYSIS OR COLLOID CHEMISTRY; THEIR RELEVANT APPARATUS
- B01J20/00—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof
- B01J20/22—Solid sorbent compositions or filter aid compositions; Sorbents for chromatography; Processes for preparing, regenerating or reactivating thereof comprising organic material
- B01J20/24—Naturally occurring macromolecular compounds, e.g. humic acids or their derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- Organic Chemistry (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Biochemistry (AREA)
- Biophysics (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Medicinal Chemistry (AREA)
- Molecular Biology (AREA)
- Gastroenterology & Hepatology (AREA)
- Zoology (AREA)
- Toxicology (AREA)
- Analytical Chemistry (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Peptides Or Proteins (AREA)
Abstract
The invention provides a pentapeptide, wherein the amino acid sequence of the pentapeptide is as follows: Lys-Phe-Phe-Ser-Cys. The pentapeptide can be specifically combined with serum albumin, and can be used for separating, purifying or eliminating the serum albumin from a mixture.
Description
Technical Field
The invention belongs to the technical field of biology, and particularly relates to pentapeptide and application thereof.
Background
Serum albumin is the most abundant protein in vertebrate plasma, and has physiological functions of combining and transporting endogenous and exogenous substances, maintaining blood osmotic pressure, eliminating free radicals, inhibiting platelet aggregation, resisting blood coagulation and the like. The amino acid sequence of serum albumin is well conserved, and serum albumin from different sources has similar spatial structure. The serum albumin is widely applied to the fields of biology, medicine, chemical industry and the like, and has considerable social and economic benefits. At present, the serum albumin is mostly separated by salting out and ion exchange chromatography, the separation process is complex, and the separation effect is unsatisfactory. Therefore, the separation and purification technology of serum albumin still needs to be improved.
Affinity separation is a separation and purification method established by utilizing the characteristics of specific recognition and reversible binding of certain affinity ligands to biological macromolecules, and certain protein with high purity can be obtained by only one-step treatment. The affinity ligand is an affinity separation core, so the development of the serum albumin affinity ligand is very important for improving the separation and purification technology of the serum albumin.
Disclosure of Invention
In order to solve the problems in the prior art, the present invention provides a pentapeptide, which can specifically bind to serum albumin, thereby separating, purifying or eliminating the serum albumin from a mixture.
The invention provides a pentapeptide, wherein the amino acid sequence of the pentapeptide is as follows: Lys-Phe-Phe-Ser-Cys.
The invention also provides the application of the pentapeptide in the specific binding of serum albumin.
Compared with the prior art, the invention has the following advantages and beneficial effects:
1) high efficiency: the serum albumin adsorbing peptide can be specifically combined with the serum albumin.
2) Safety: the serum albumin has short adsorption peptide sequence, no immunogenicity, safety and no toxicity.
3) Economy: the serum albumin adsorption peptide is obtained by solid phase synthesis, and the technology is mature.
4) And the thiol at the Cys end realizes directional crosslinking, and the medium is simple to prepare.
Therefore, the serum albumin adsorption peptide of the invention is used as an affinity ligand, and the separation, purification or elimination of the serum albumin from the mixture can be realized.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
EXAMPLE 1 polypeptide Synthesis
1) Activating resin: 1000mg of Fmoc Cys-wang Resin was weighed and soaked in DMF for 30min for swelling.
2) Deprotection: filtering to remove DMF, adding 20% piperidine-containing DMF solution, blowing to boil for 20min, and filter pressing to remove DMF; washing the resin with isopropanol three times, washing the resin with DMF three times, and detecting the Fmoc removal condition of the resin by an ninhydrin method.
3) Condensation reaction: connecting amino acid Ser, weighing 1.4mmol of FMOC-Ser, taking a DMF mixed solution of TBTU/HOBt/DIEA as a reaction solution, and blowing nitrogen at room temperature for reaction for 3 h. After the reaction, the resin is washed by isopropanol three times, then washed by DMF three times, and respectively filtered and removed, and detected by an indantrione method. Fmoc synthesis starts at the C-terminus and ends at the N-terminus.
4) Repeating steps 2) to 3): and sequentially connecting Ser, Phe and Lys to complete polypeptide chain extension. The polypeptide from N-terminus to C-terminus is as follows: Lys-Phe-Phe-Ser-Cys.
5) Polypeptide cleavage: blowing the polypeptide-resin composite with nitrogen gasphenol/H2And (3) taking TFA mixed solution of O/thioanisole/EDT/TIS as a cutting reagent, magnetically stirring for 3 hours, directly dripping the suction filtration solution into frozen ether, centrifuging at 3000r/min, collecting precipitate, and freeze-drying to constant weight to obtain the crude peptide.
6) Purifying the crude peptide to be more than 95% by HPLC, wherein the purification condition is to re-dissolve the crude peptide in 10% acetonitrile solution, take acetonitrile as a mobile phase, perform gradient elution for 30 minutes by adopting 10% to 100% acetonitrile mobile phase, collect the largest peak, obtain the purified polypeptide, and the polypeptide sequence is as follows: Lys-Phe-Phe-Ser-Cys, polypeptide purity 97%.
Example 2 affinity media preparation
1) 10mg of the polypeptide prepared in step 1 was dissolved in 20mL of Hepes buffer, pH 7.0.
2) 2mL of Sephadex gel after thiol activation by GE was collected.
3) The two were mixed and reacted for 4 hours.
4) After the reaction, the gel particles were washed with Hepes buffer to obtain the affinity medium.
Example 3 separation and purification of serum Albumin by serum Albumin adsorption peptide
1) 10mmol/L Hepes buffer solution with pH7.0 and NaCl content 0.15M is prepared. The flow rate was set at 1 mL/min.
2) And (3) operating an AKTA protein purification system by taking the liquid prepared in the step 1) as a mobile phase.
3) The column was packed with 5mL of the affinity medium of example 2.
4) 10mL of serum albumin to be separated liquid is respectively loaded.
5) 0.1M HCl-Gly buffer at pH2 was prepared as eluent.
6) And (4) loading 15mL of eluent, and collecting the eluent.
7) And respectively measuring the content and purity of the serum albumin in the liquid to be separated of the serum albumin and the eluent, and calculating the extraction rate.
TABLE 1 adsorption characteristics of serum albumin adsorbing peptide media
As is clear from Table 1, the serum albumin adsorbing peptide of the present invention can achieve the separation, purification or removal of serum albumin from a mixture.
Example 4 affinity media preparation
1) 10mg of the polypeptide prepared in step 1 was dissolved in 20mL of Hepes buffer, pH 7.0.
2) 2mL of thiol-activated Sepharose gel from GE was collected.
3) The two were mixed and reacted for 4 hours.
4) After the reaction, the gel particles were washed with Hepes buffer to obtain the affinity medium.
Example 5 serum Albumin removal by serum Albumin adsorbing peptides
1) 10mmol/L Hepes buffer solution with pH7.0 and NaCl content 0.15M is prepared. The flow rate was set at 1 mL/min.
2) And (3) operating an AKTA protein purification system by taking the liquid prepared in the step 1) as a mobile phase.
3) The column was packed with 5mL of the affinity medium of example 4.
4) 10mL of the mixed solution of serum albumin and hemoglobin is loaded.
5) 0.1M HCl-Gly buffer at pH2 was prepared as eluent.
6) And (4) loading 15mL of eluent, and collecting the eluent.
7) The eluent is put into an ultrafiltration centrifugal tube with the molecular weight cutoff of 30000, and is centrifuged for 10min at 5000rpm, and the residual eluent is collected.
8) Repeating the steps 4), 5) and 6) once.
7) And respectively measuring the content and purity of the serum albumin and the hemoglobin in the mixed solution of the serum albumin and the hemoglobin and the eluent, and calculating the total clearance rate.
TABLE 2 adsorption characteristics of serum albumin adsorption peptide media
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the 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> south Yang college of learning
<120> pentapeptide and application thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 5
<212> PRT
<213> Artificial Sequence (Artificial Sequence)
<400> 1
Lys Phe Phe Ser Cys
1 5
Claims (2)
1. A pentapeptide, characterized by: the amino acid sequence of the pentapeptide is as follows: Lys-Phe-Phe-Ser-Cys.
2. Use of the pentapeptide of claim 1 for specifically binding serum albumin; the pentapeptide is applied to the separation and purification of serum albumin or the elimination of serum albumin for the purposes of diagnosis and treatment of non-diseases.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811311659.3A CN109608518B (en) | 2018-11-06 | 2018-11-06 | Pentapeptide and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201811311659.3A CN109608518B (en) | 2018-11-06 | 2018-11-06 | Pentapeptide and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN109608518A CN109608518A (en) | 2019-04-12 |
| CN109608518B true CN109608518B (en) | 2022-04-29 |
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| Application Number | Title | Priority Date | Filing Date |
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| CN201811311659.3A Active CN109608518B (en) | 2018-11-06 | 2018-11-06 | Pentapeptide and application thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110818776B (en) * | 2019-12-04 | 2022-07-12 | 南阳师范学院 | Affinity peptide and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102781959A (en) * | 2010-02-05 | 2012-11-14 | 埃博灵克斯股份有限公司 | Peptides capable of binding to serum albumin and compounds, constructs and polypeptides comprising the same |
| CN103897031A (en) * | 2012-12-25 | 2014-07-02 | 深圳先进技术研究院 | Chemically modified thymopentin and synthetic method thereof |
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2018
- 2018-11-06 CN CN201811311659.3A patent/CN109608518B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102781959A (en) * | 2010-02-05 | 2012-11-14 | 埃博灵克斯股份有限公司 | Peptides capable of binding to serum albumin and compounds, constructs and polypeptides comprising the same |
| CN103897031A (en) * | 2012-12-25 | 2014-07-02 | 深圳先进技术研究院 | Chemically modified thymopentin and synthetic method thereof |
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| CN109608518A (en) | 2019-04-12 |
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