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Large-scale preparation method and application of polypeptide
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A large-scale, synthetic method technology, applied in the field of medicine and biology, can solve problems such as the inability to achieve large-scale synthesis
Pending Publication Date: 2020-06-30
THE NAT CENT FOR NANOSCI & TECH NCNST OF CHINA
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[0003] E5 polypeptide is a cancer-targeting polypeptide, which has a strong ability to inhibit cancercell migration and invasion. E5 can provide feasible methods and technologies for inhibiting cancermetastasis and treating cancer. However, due to its long amino acid sequence, and sulfhydryl and Due to the presence of many hydrophobic amino acids, conventional solid-phase synthesis methods cannot achieve large-scale synthesis of them
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Embodiment 1
[0110] This example is used to illustrate the method for large-scale synthesis of polypeptides of the present invention.
[0111] This example provides a large-scale synthesis method of E5 polypeptide.
[0112] The amino acid sequence of the E5 polypeptide is: GGRSSFFLLRRIQGCFRRNTVDD
[0113] 1. Synthetic peptide chain
[0114] 1) Link the first amino acid
[0115] Get 400g 2-chlorotriphenyl chloride resin, replace 0.938mmol / g, soak 30 minutes with DCM in the solid-state synthesis reactor, make resin fully swell, use vacuum water pump to drain liquid, add 159g Fmoc-Asp(OtBu)- OH amino acid and 4LDMF, react for 30min.
[0116] Drain the liquid, wash the resin alternately with 4LDMF and DCM three times, and then wash with 4LDMF three times. Drain the liquid. Add 8 L of DCM, 1.5 L of methanol, and 0.5 L of DIEA mixed solution to block the chlorine on the unreacted resin, and react for 10 min. repeat. Wash with 4L DMF and DCM alternately for 3 times, and then wash with 4L D...
Embodiment 2
[0176] This example is used to illustrate the method for large-scale synthesis of polypeptides of the present invention.
[0177] This example provides a method for large-scale synthesis of polypeptides.
[0178] The amino acid sequence of the CD36-6 polypeptide is: FITC-RGVYDVFNGDRNISD
[0179] 1. Synthetic peptide chain
[0180] 1) Link the first amino acid
[0181] Get 400g 2-chlorotriphenyl chloride resin, replace 0.938mmol / g, soak 30 minutes with DCM in the solid-state synthesis reactor, make resin fully swell, use vacuum water pump to drain liquid, add 159g Fmoc-Asp(OtBu)- OH amino acid and 4LDMF, react for 30min.
[0182] Drain the liquid, wash the resin alternately with 4LDMF and DCM three times, and then wash with 4LDMF three times. Drain the liquid. Add 8 L of DCM, 1.5 L of methanol, and 0.5 L of DIEA mixed solution to block the chlorine on the unreacted resin, and react for 10 min. repeat. Wash with 4L DMF and DCM alternately for 3 times, and then wash with 4...
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Abstract
The invention provides a large-scale preparation method and application of polypeptide. The method comprises the following steps of: taking 2-chloro-triphenylchloride resin as a starting raw material,connecting a first Fmoc protective amino acid, and sealing by using a sealing reagent; taking the Fmoc protective amino acid as a monomer, taking a hexahydropyridine solution as a deprotection reagent, and sequentially connecting amino acids one by one under the action of a condensing agent and an alkaline condition to synthesize the polypeptide; cutting the polypeptide, and concentrating to obtain a polypeptide crude product; and purifying the crude product to obtain a polypeptide product. The method can be used for preparing pure peptides of a hectogram grade and a kilogram grade, the problem of gram-scale production of the polypeptide as a raw materialmedicine is solved, and a simple and effective way is provided for preparing a reaction biological material.
Description
technical field [0001] The invention belongs to the field of medical biotechnology, and in particular relates to a large-scale preparation method and application of a polypeptide. Background technique [0002] The chemical synthesis of peptides is divided into two methods: liquid phase and solid phase. The liquid phase method is suitable for the synthesis of small peptides. For long peptides, the solidphase method is currently the most commonly used peptide synthesis method. The principle of solid-phase peptide synthesis reaction is to fix the carboxyl group of the first amino acid at the C-terminal of the polypeptide sequence on an incompatible carrier (resin) through condensation reaction, then remove the amino protecting group, and then combine with the excess activated first amino acid The carboxyl groups of two amino acids react to form a peptide bond. Repeat the deprotection, condensation, and washing processes to extend the peptide chain in sequence, and finally obt...
Claims
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