Process for folding chemically synthesized polypeptides

A technology for chemical synthesis and polymerization of carriers, which is used in the preparation methods of peptides, chemical instruments and methods, organic chemistry, etc.

Inactive Publication Date: 2003-10-15
RMF DICTAGENE
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

These observations strongly suggest that changes in the primary structure of polysulfhydryl precursors may adversely affect local correct folding (β-turns, polyproline helical motifs, etc.) in the polypeptide chain to be folded

Method used

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  • Process for folding chemically synthesized polypeptides
  • Process for folding chemically synthesized polypeptides
  • Process for folding chemically synthesized polypeptides

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0079] Example 1 Cys 10，11，34，50 (S-t-Bu)-hu-TARC (thymus and activation-regulated chemokine) synthesis and fold

[0080] Chemokine derivatives with 71 amino acid residues were assembled onto a 433A peptide synthesizer (Perkin Elmer / ABI) using Fmoc / t-Bu chemistry and polystyrene-based resins that were acid-resistant Functionalized with a stable hydroxymethylphenoxyacetic acid linker (Wang resin), Fmoc-Ser(t-Bu) was linked to the on the resin. The degree of substitution is 0.57 mmole / g. The synthesis reaction was carried out on a scale of 0.27 mmole by utilizing a five-fold excess of Fmoc-amino acid and DCI (N, N'-diisopropylcarbodiimide) / HOBt (1-hydroxybenzotriazole) activating reagent in performed in DMF. The coupling reaction time was approximately 60 minutes under conditions where Fmoc deprotection was monitored spectrophotometrically.

[0081] The four cysteine ​​sulfhydryl groups were protected with S-tert-butyl groups and the maximal protection scheme was used...

Embodiment 2

[0089] Example 2 Cys 10，34，50 (S-t-Bu)-hu-TARC and Cys 11，34，50 Synthesis of (S-t-Bu)-hu-TARC and fold

[0090] Cys 10，34，50 (S-t-Bu)hu-TARC and Cys 11，34，50 The conditions used for the synthesis, purification and folding of (S-t-Bu)-hu-TARC derivatives and Cys 10，11，34，50 The conditions adopted by (S-t-Bu)hu-TARC (Example 1) are the same, the only difference is that Cys 10 and Cys 11 Trt protection was performed, and the protecting group was removed at the same time as the cleavage of the polypeptide precursor from the resin. The yields of the final folded chemokines were 80% and 79%, respectively.

Embodiment 3

[0091] Example 3 CVs 34，50 Synthesis and Folding of (S-Bu)-hu-TARC

[0092] In addition to using Trt to Cys 10 and Cys 11 In addition to the protection, the Cys 34，50The conditions adopted for the synthesis, purification and folding of (S-Bu)-hu-TARC derivatives are the same as the conditions adopted for the derivatives of Example 1 and Example 2. is removed. The yield of folded product was about 75%.

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Abstract

The present invention relates to a process for folding chemically synthesized polypeptides, comprising treating a polypeptide and / or protein that comprises two or more derivatized cysteine residues with a reducing agent in a folding buffer having a predetermined pH and temperature, wherein the derivatized cysteine residue corresponds to S-butyl-thio-cysteine residue and wherein the reducing agent is cysteine.

Description

technical field [0001] The invention relates to a method for folding chemically synthesized polypeptides. In addition, the present invention also relates to a method for producing biologically active protein. Background technique [0002] Demand for synthetic proteins has gradually increased in recent decades since the successful chemical synthesis of fully active HIV-protease prepared by highly optimized solid-phase peptide synthesis (SPPS) A 99-residue enzyme, the method is based on the standard Boc / Bzl method. [0003] The synthesis of crystalline ubiquitin in 1994 further showed that high-purity proteins can be synthesized by the SPPS method based on the Fomc / t-Bu scheme, which is simpler in operation than the Boc / Bzl method and chemically inferior to the Boc / Bzl method complex, wherein the crystalline ubiquitin protein is a small molecule protein consisting of 76 residues. [0004] By 2000, substantial experimental evidence demonstrated that single-domain proteins co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K1/04C07K1/113C07K1/14
CPCC07K1/04C07K1/1133C07K1/113
Inventor A·瓦尔狄尼G·克拉丁M·洛戈罗
Owner RMF DICTAGENE
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