A method for splitting thymosin β4

Abstract

The invention discloses a method for pyrolysis of thymosin beta-4. The method comprises the step of carrying out pyrolysis on thymosin beta-4 peptide resin to obtain crude peptide, wherein a mixed solvent of trifluoroacetic acid, thioanisole, dithioglycol, phenol, water, dimethyl sulfide and ammonium iodide is used as a pyrolysis reagent in the pyrolysis process. According to the method for pyrolysis of thymosin beta-4, the purity of crude peptide is greatly improved, meanwhile the total yield is greatly increased, the production cost is reduced, and the method is simple in process and convenient for industrial production.

Description

technical field

[0001] The invention relates to a method for cleavage of polypeptides, in particular to a method for cleavage of thymosin β4. Background technique

[0002] Thymosin was first extracted from calf thymus by Goldstein and White in 1966. According to the different isoelectric points, thymosin can be divided into 3 types: α (PI<5), β (5<PI<7) and γ (PI>7). Constructs have been identified, and there are three main types in the human body, namely thymosin β4 (Tβ4), Tβ10 and Tβ15, of which thymosin β4 has the highest content. Thymosin β4 is a highly conserved water-soluble polypeptide composed of 43 amino acid residues, with a relative molecular mass of 4964.

[0003] The molecular formula of thymosin β4 is:

[0004] Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu -Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser-OH Thymosin β4 is considered to be predominantly spherical Ac...

Examples

Embodiment 1

[0063] Thymosin β4 peptide resin was obtained by solid phase synthesis.

[0064] Prepare a lysate with a mass ratio (trifluoroacetic acid: thioanisole: ethanedithiol: phenol: water = 86:4:3:4:3) of 100 grams, and stir the prepared lysate evenly. Weigh 5% thymosin β4 peptide resin (5 g), add to the above lysate, and react for 120 minutes. After the reaction is completed, filter the above reaction solution with a sand core funnel, collect the filtrate, wash the filter cake with trifluoroacetic acid, and combine the filtrate. The filtrate was added to 1000 ml of glacial ether for precipitation, filtered with a sand core funnel, the filter cake was collected, washed with glacial ether, and put into a vacuum drying oven for vacuum drying to obtain thymosin β4 crude peptide. Weighed to get 2.55 grams of crude product, crude peptide weight yield: 96.2%, through HPLC detection, crude peptide purity: 29.3%

[0065] Purified by high performance liquid phase to obtain thymosin β4 fine ...

Embodiment 2

[0067] Thymosin β4 peptide resin was obtained by solid phase synthesis.

[0068] Prepare lysate, mass ratio (trifluoroacetic acid: thioanisole: ethanedithiol: phenol: water: methyl sulfide = 84: 4: 3: 4: 3: 2) 100 grams, stir the prepared lysate uniform. Weigh 5% thymosin β4 peptide resin (5 g), add to the above lysate, and react for 120 minutes. After the reaction is completed, filter the above reaction solution with a sand core funnel, collect the filtrate, wash the filter cake with trifluoroacetic acid, and combine the filtrate. The filtrate was added to 1000 ml of glacial ether for precipitation, filtered with a sand core funnel, the filter cake was collected, washed with glacial ether, and put into a vacuum drying oven for vacuum drying to obtain thymosin β4 crude peptide. Weighed 2.63 grams of crude product, crude peptide weight yield: 99.2%, through HPLC detection, crude peptide purity: 33.5%

[0069] Purified by high performance liquid phase to obtain thymosin β4 fi...

Embodiment 3

[0071] Thymosin β4 peptide resin was obtained by solid phase synthesis.

[0072] Configure lysate, mass ratio (trifluoroacetic acid: thioanisole: ethanedithiol: phenol: water: methyl sulfide: ammonium iodide = 83.5: 4: 3: 4: 3: 2: 0.5) 100 grams, will The prepared lysate was stirred evenly. Weigh 5% thymosin β4 peptide resin (5 g), add to the above lysate, and react for 120 minutes. After the reaction is completed, filter the above reaction solution with a sand core funnel, collect the filtrate, wash the filter cake with trifluoroacetic acid, and combine the filtrate. The filtrate was added to 1000 ml of glacial ether for precipitation, filtered with a sand core funnel, the filter cake was collected, washed with glacial ether, and put into a vacuum drying oven for vacuum drying to obtain thymosin β4 crude peptide. Weighed 2.88 grams of crude product, crude peptide weight yield: 108.7%, tested by HPLC, crude peptide purity: 43.5%

[0073] Purified by high performance liquid ...

technical field

[0001] The invention relates to a method for cleavage of polypeptides, in particular to a method for cleavage of thymosin β4. Background technique

[0002] Thymosin was first extracted from calf thymus by Goldstein and White in 1966. According to the different isoelectric points, thymosin can be divided into 3 types: α (PI<5), β (5<PI<7) and γ (PI>7). Constructs have been identified, and there are three main types in the human body, namely thymosin β4 (Tβ4), Tβ10 and Tβ15, of which thymosin β4 has the highest content. Thymosin β4 is a highly conserved water-soluble polypeptide composed of 43 amino acid residues, with a relative molecular mass of 4964.

[0003] The molecular formula of thymosin β4 is:

[0004] Ac-Ser-Asp-Lys-Pro-Asp-Met-Ala-Glu-Ile-Glu-Lys-Phe-Asp-Lys-Ser-Lys-Leu-Lys-Lys-Thr-Glu-Thr-Gln-Glu -Lys-Asn-Pro-Leu-Pro-Ser-Lys-Glu-Thr-Ile-Glu-Gln-Glu-Lys-Gln-Ala-Gly-Glu-Ser-OH Thymosin β4 is considered to be predominantly spherical Ac...

Examples

Embodiment 1

[0063] Thymosin β4 peptide resin was obtained by solid phase synthesis.

[0064] Prepare a lysate with a mass ratio (trifluoroacetic acid: thioanisole: ethanedithiol: phenol: water = 86:4:3:4:3) of 100 grams, and stir the prepared lysate evenly. Weigh 5% thymosin β4 peptide resin (5 g), add to the above lysate, and react for 120 minutes. After the reaction is completed, filter the above reaction solution with a sand core funnel, collect the filtrate, wash the filter cake with trifluoroacetic acid, and combine the filtrate. The filtrate was added to 1000 ml of glacial ether for precipitation, filtered with a sand core funnel, the filter cake was collected, washed with glacial ether, and put into a vacuum drying oven for vacuum drying to obtain thymosin β4 crude peptide. Weighed to get 2.55 grams of crude product, crude peptide weight yield: 96.2%, through HPLC detection, crude peptide purity: 29.3%

[0065] Purified by high performance liquid phase to obtain thymosin β4 fine ...

Embodiment 2

[0067] Thymosin β4 peptide resin was obtained by solid phase synthesis.

[0068] Prepare lysate, mass ratio (trifluoroacetic acid: thioanisole: ethanedithiol: phenol: water: methyl sulfide = 84: 4: 3: 4: 3: 2) 100 grams, stir the prepared lysate uniform. Weigh 5% thymosin β4 peptide resin (5 g), add to the above lysate, and react for 120 minutes. After the reaction is completed, filter the above reaction solution with a sand core funnel, collect the filtrate, wash the filter cake with trifluoroacetic acid, and combine the filtrate. The filtrate was added to 1000 ml of glacial ether for precipitation, filtered with a sand core funnel, the filter cake was collected, washed with glacial ether, and put into a vacuum drying oven for vacuum drying to obtain thymosin β4 crude peptide. Weighed 2.63 grams of crude product, crude peptide weight yield: 99.2%, through HPLC detection, crude peptide purity: 33.5%

[0069] Purified by high performance liquid phase to obtain thymosin β4 fi...

Embodiment 3

[0071] Thymosin β4 peptide resin was obtained by solid phase synthesis.

[0072] Configure lysate, mass ratio (trifluoroacetic acid: thioanisole: ethanedithiol: phenol: water: methyl sulfide: ammonium iodide = 83.5: 4: 3: 4: 3: 2: 0.5) 100 grams, will The prepared lysate was stirred evenly. Weigh 5% thymosin β4 peptide resin (5 g), add to the above lysate, and react for 120 minutes. After the reaction is completed, filter the above reaction solution with a sand core funnel, collect the filtrate, wash the filter cake with trifluoroacetic acid, and combine the filtrate. The filtrate was added to 1000 ml of glacial ether for precipitation, filtered with a sand core funnel, the filter cake was collected, washed with glacial ether, and put into a vacuum drying oven for vacuum drying to obtain thymosin β4 crude peptide. Weighed 2.88 grams of crude product, crude peptide weight yield: 108.7%, tested by HPLC, crude peptide purity: 43.5%

[0073] Purified by high performance liquid ...

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