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Novel method for oxidative folding of protein

A protein and oxidant technology, applied in chemical instruments and methods, peptide preparation methods, organic chemistry, etc., to reduce production costs, facilitate production operations, and simple ingredients

Inactive Publication Date: 2011-09-28
WEIZHI LANSI MEDICAL TECH BEIJING
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, there is no direct evidence on whether various chromatographic matrices can directly promote protein refolding [Jungbauer A, Kaar W, Schlegl R. Folding and refolding of proteins inchromatographic beds. Curr Opin Biotechnol. 2004; 15: 487-94 .]

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  • Novel method for oxidative folding of protein
  • Novel method for oxidative folding of protein
  • Novel method for oxidative folding of protein

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Embodiment Construction

[0030] The statements of the following embodiments are for better understanding of the principle and application of the present invention, but not to limit the application scope of the present invention.

[0031] Materials and Methods:

[0032] 1. Protein sample: bovine pancreatic RNase A was purchased from Beijing Qinyuan Huizhi Biotechnology Co., Ltd.; electrophoresis and mass spectrometry analysis conformed to its theoretical molecular weight (13689.33). The cDNA encoding human pancreatic RNase I was amplified from the reverse transcription product of total RNA of human pancreatic cancer cell PANC-1 by PCR reaction. The gene encodes a mature human pancreatic RNase I protein of 128 amino acid residues. Using the gene as a template, primers are designed to amplify gene fragments encoding S peptide (1-15 amino acid sequence) and S protein (21-128 amino acid sequence). A mutant gene encoding S protein C-terminal fusion S peptide was generated by enzyme-cleavage ligation. The...

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Abstract

The invention discloses a novel method for oxidative folding of a protein, wherein the protein contains at least one disulfide bond when the protein is in an active state. The method comprises changing the state of the protein from a denatured state to an active state so as to obtain bioactive protein products. In the method, through a contact and / or the adsorption between a chromatographic matrix and a denatured protein, a denaturant and other impurities are separated from the denatured protein, and thus the denatured protein or / and a composition intermediate thereof form rapidly a stable intermediate with a nearly natural structure, wherein the stable intermediate can be treated only by an oxidizing agent. Through utilizing the method, formations of disulfide bonds with correct conformations in protein molecules are promoted and active protein products can be obtained under a wide range of reaction conditions.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and relates to the renaturation technology of denatured protein, mainly the oxidative renaturation technology. Background technique: [0002] Recombinant DNA technology makes it possible to express polypeptides and protein molecules in large quantities in bacteria or other host cells. Recombinantly expressed protein products have had many successes. However, heterologous host expression technology often encounters the situation that the protein product is precipitated in the host cell. Precipitated proteins exist in the form of inclusion bodies, which do not have a natural three-dimensional structure, are in a denatured state, and usually have no biological activity. [0003] Denatured proteins need to be processed in vitro to obtain a biologically active spatial structure. This process is called renaturation or protein folding (refolding). In vitro renaturation is often a key factor in the succ...

Claims

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Application Information

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IPC IPC(8): C07K1/00
Inventor 王楠芦娜吕春婉
Owner WEIZHI LANSI MEDICAL TECH BEIJING
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