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Method for efficient expression, renaturation and purification of lipocalin

A protein and soluble technology, applied in the fields of high-efficiency expression, purification and renaturation of lipocalin

Inactive Publication Date: 2012-02-01
SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how to denature and renature inclusion bodies and obtain proteins with good biological activity after renaturation is difficult for those skilled in the art.

Method used

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  • Method for efficient expression, renaturation and purification of lipocalin
  • Method for efficient expression, renaturation and purification of lipocalin
  • Method for efficient expression, renaturation and purification of lipocalin

Examples

Experimental program
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Effect test

preparation example Construction

[0041] The method for preparing soluble lipocalin of the present invention comprises the following steps:

[0042] (1) denature the inclusion body of lipocalin, and separate the supernatant containing denatured lipocalin;

[0043] (2) Adjust the pH value of the obtained supernatant to 5-6, and perform renaturation with PEG with a degree of polymerization of 200-1000 and polyhydric alcohols of C2-C8 to obtain soluble lipocalin.

[0044] protein expression

[0045] The present inventors tried to express lipocalin by adopting various expression systems (such as yeast system and Escherichia coli system), and found that the Escherichia coli system is more suitable for expressing lipocalin and obtaining the protein in the form of inclusion bodies.

[0046]Studies have shown that the main reason for the formation of inclusion bodies is that the protein expression rate is too fast, so that the protein does not have enough time to fold correctly; in addition, because the cytoplasmic e...

Embodiment 1

[0094] Example 1. Expression of rLcn 8 in Escherichia coli BL21 (DE3)

[0095] The expression plasmid pET28b-L was transformed into BL21(DE3) competent cells, and positive transformants were obtained by colony PCR and sequence determination. The transformants were cultured with shaking at 37°C in LB containing 50 μg / ml kanamycin. When the body density OD600 was 0.5, 1 mmol / L IPTG was added to induce the expression of the target protein.

[0096] After 5h, the bacteria were collected by centrifugation at 7000g for 5min, and the bacteria were crushed with 50ml of 50mmol / L Tris-HCl pH 8.0, 100mmol / L NaCl, 5% (v / v) glycerol mixture, and after centrifugation at 12000rpm for 20min, the precipitate and the above Clear, 15% SDS-PAGE analysis of expression.

[0097] The rLcn 8 constructed in this study has a total of 182 amino acids and a theoretical molecular weight of 20847.3D. From the electrophoresis results ( figure 1 ), the rLcn 8 protein band was located near 20.1kD, which was...

Embodiment 2

[0098] Example 2. Washing and denaturation of inclusion bodies

[0099] Resuspend the precipitate obtained above with 1 mol / L urea, homogenize with a homogenizer, supplement with short-term ultrasonic pulverization, centrifuge at 12,000 rpm for 10 min, discard the supernatant, and repeat the above process once.

[0100] Inclusion body with 50mmol / L NaH 2 PO 4 , 5mol / L guanidine hydrochloride, mixed overnight at 4°C, and centrifuged at 12000rpm for 30min to remove the precipitate.

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Abstract

The invention relates to a method for efficient expression, renaturation and purification of lipocalin. The yield and purity of soluble protein are effectively improved via adjustment of the specific pH value at a specific stage and selection of appropriate denaturants and renaturation agents. Moreover, the natural structure of the prepared soluble protein can be better restored, and the biological activity of the prepared soluble protein can be better maintained.

Description

technical field [0001] The invention belongs to the field of biotechnology; more specifically, the invention relates to a method for high-efficiency expression, renaturation and purification of lipocalin. Background technique [0002] The lipocalin family is composed of small secreted proteins, and its members are found in vertebrates, invertebrates, plants and bacteria, and the size is generally 120-180 amino acids. On the primary sequence, lipocalins all contain short conserved domains [Flower DR, North AC, Attwood TK. Structure and sequence relationships in the lipocalins and related proteins. Protein Sci. 1993May; 2(5): 753-61; Flower DR, North AC, Attwood TK. Mouse oncogene protein 24p3 ​​is a member of the lipocalin protein family. Biochem Biophys Res Commun. 1991 Oct 15; 180 (1): 69-74], their tertiary structure usually contains 6 - or the 8-strand β-sheet forms a barrel structure [George Kontopidis, Carl Holt and Lindsay Sawyer. The Ligand-binding Site of Bovine β-L...

Claims

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Application Information

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IPC IPC(8): C07K14/00C07K1/14C12N15/11C12N15/70
Inventor 林胜祥陈泽庸邹永水
Owner SHANGHAI INST OF BIOLOGICAL SCI CHINESE ACAD OF SCI
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