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Method for producing recombinant protein A

A technology of recombinant protein and protein, which is applied in the field of high-density fermentation and protein separation and purification, to achieve the effects of low production cost, easy amplification, and high binding activity

Active Publication Date: 2010-12-22
DALIAN UNIV OF TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Aiming at the current situation of large market demand for protein A and lack of low-cost, high-quality large-scale production technology, we propose a new method for large-scale production of recombinant protein A, including high-density cultivation of genetically engineered strains and recombinant protein A separation and purification method

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  • Method for producing recombinant protein A
  • Method for producing recombinant protein A
  • Method for producing recombinant protein A

Examples

Experimental program
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Embodiment 1

[0025] 1. Shake flask culture of genetically engineered bacteria

[0026] The genetically engineered bacteria expressing protein A were inoculated in 20 mL of LB liquid medium, and cultured overnight in a constant temperature shaker at 37° C. and 200 rpm. Inoculate the activated bacteria in 100 mL LB liquid medium according to the inoculum amount of 1%. Cultivate at 37°C, 200rpm for 3-4 hours, until the cell OD 600nm When it reaches 0.5-1, add IPTG to 0.2mM or add lactose to 1g / L, and continue to cultivate for 5-7 hours. SDS-PAGE detection confirmed the expression of recombinant protein A.

[0027] 2. High-density fermentation expression of recombinant protein A

[0028] (1) Configuration of fermentation medium

[0029] After sterilizing glucose (or glycerol), peptone, yeast extract, inorganic salts, and trace elements respectively, prepare a basic medium with the following final concentrations: 10-20g / L peptone, 5-10g / L yeast extract, 20 -50mM Na 2 HPO 4 , 20-50mM KH ...

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Abstract

The invention relates to a method for producing recombinant protein A, which belongs to the technical field of biological engineering and provides a technological condition for fermenting and expressing the recombinant protein A in high density by using genetic engineering bacteria, and a method for separating and purifying the recombinant protein A in high purity by using Fc segment of IgG as anaffinity ligand by a one-step affinity chromatography method. By applying the method for producing the recombinant protein A, the culture density of the recombinant bacteria fermented in high densityreaches OD600nm=80-100; the dry weight of the bacteria is 40-50g / L; the expression quantity of the protein A occupies 30% to 50% of total protein of the bacteria; the quantity of the protein A produced by each liter of bacterial liquid reaches 3g; and the purity of the protein A detected by SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) and HPLC (High Performance Liquid Chromatography) is more than 95%. The method has the advantages of large yield, low cost, high product quality, and the like, and provides a practicable approach for preparing the recombinant protein A.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a high-density fermentation of genetically engineered bacteria and a method for separating and purifying proteins. Background technique [0002] Staphylococcus aureus protein A (Staphylococcus aureus Protein A, SPA) is the cell wall binding protein of certain species of Staphylococcus aureus, which was studied by Danish scientist Klaus Jensen in the 1950s to study the cell wall structure of Staphylococcus aureus discovered when. Its basic structure is composed of the following three parts, namely signal peptide, antibody binding functional region and cell wall binding region. The antibody-binding functional region of protein A contains five homologous single domains E, D, A, B, and C, and each single domain can act independently with immunoglobulin G (IgG). The binding of the protein to IgG is mainly based on the specific binding ability to the Fc region of I...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P21/00C07K1/22C12R1/19
Inventor 贾凌云林雪侯率张嘉玉任军谢键
Owner DALIAN UNIV OF TECH
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