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Efficient soluble expression and purification method of human LOX-1 (lectin like oxidized low density lipoprotein receptor-1) extracellular domain

An expression method and exogenous structure technology, applied in the field of efficient soluble expression and purification of human LOX-1 extracellular domain, can solve the problems of harsh reaction conditions, poor protein stability, time-consuming efficiency, etc., and achieve simple and efficient recovery and maintenance High stability and high purity

Inactive Publication Date: 2012-07-25
JILIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the renaturation of inclusion bodies has the following disadvantages: ①The reaction conditions are harsh and require the participation of protein denaturants; ②The process is complicated, time-consuming and inefficient; ③The recovery rate is low, and it is easy to cause a large amount of loss; restricted in practical application
Soluble expression of LOX-1 in Escherichia coli has not been reported

Method used

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  • Efficient soluble expression and purification method of human LOX-1 (lectin like oxidized low density lipoprotein receptor-1) extracellular domain
  • Efficient soluble expression and purification method of human LOX-1 (lectin like oxidized low density lipoprotein receptor-1) extracellular domain
  • Efficient soluble expression and purification method of human LOX-1 (lectin like oxidized low density lipoprotein receptor-1) extracellular domain

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] Example 1 : Construction of expression vector and verification of soluble expression

[0055] 1. Construction of expression vector

[0056] Select the hLOX-1 ECD domain as the expression object. The hLOX-1 ECD gene fragment was amplified from human LOX-1 cDNA by PCR reaction. Gateway technology is used to connect the target gene with the expression vector.

[0057] figure 2 is the model diagram of the constructed expression vector, wherein, MBP represents maltose binding protein; Thrombin site represents the thrombin cleavage site.

[0058] Specific steps are as follows:

[0059] 1. Using human LOX-1 cDNA as a template, design primers:

[0060] Primer 1: 5'-cacc CTGGTGCCACGCGGTTCT TCCCAGGTGTCTGACCTCC-3'

[0061] (The lowercase part is the guide base of the plasmid constructed by Gateway technology, and the underline indicates the thrombin recognition site)

[0062] Primer 2: 5'-tcattaCTGTGCTCTTAGGTTTGCCTTC-3'

[0063] (The lowercase part is the double s...

Embodiment 2

[0083] Example 2 : Establishment of large-scale expression and purification methods

[0084] Using the successfully constructed expression vector in Example 1, the expression conditions were optimized, and a purification method for the expressed protein was established.

[0085] The specific implementation plan is as follows:

[0086] 1. The plasmid pDMhLOX-1E was transformed into Escherichia coli Rosetta-gami2(DE3) by chemical transformation, expanded and stored for later use.

[0087] 2. Referring to Example 1, the expression conditions were optimized, and the culture volume was expanded to 1 liter. The specific implementation is as follows:

[0088] ⑴ Use a 250ml Erlenmeyer flask for pre-culture, with a liquid volume of 50ml, insert Escherichia coli and culture at 37 degrees to OD 600 = 0.8.

[0089] (2) Inoculate 3% of the inoculum into a 3 L shake flask containing 1 liter of medium, and continue to culture at 37 degrees until OD 600 = 0.5, followed by variable ...

Embodiment 3

[0108] Example 3 : Mass expression and purification of hLOX-1 ECD protein

[0109] On the basis of Examples 1 and 2, the feasibility of the invention for mass production of hLOX-1 ECD protein was verified by using a 5-liter fermenter.

[0110] The specific implementation plan is as follows:

[0111] 1. Fill 100 ml medium in a 500 ml Erlenmeyer flask as primary culture. Cultivate at 37 degrees to make the cell concentration reach OD 600 = 0.9.

[0112] 2. Inoculate 3% of the inoculum into a fermenter containing 2 liters of medium, stir at 120 rpm, adjust dissolved oxygen to 20%, and continue culturing at 37 degrees until OD 600 = 0.5-0.6. Then lower the temperature to 30 degrees, add IPTG with a final concentration of 300 micromolar to the culture medium, control the dissolved oxygen at 8-10%, and continue to cultivate until the cell concentration reaches OD 600 = 1.0-1.2, stop the fermentation and recover the bacteria.

[0113] 3. Purify the expressed hLOX-1 ECD p...

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Abstract

The invention discloses an efficient soluble expression and purification method of human LOX-1 (lectin like oxidized low density lipoprotein receptor-1) extracellular domain, and relates to construction of an expression vector for efficient soluble expression of human LOX-1 extracellular domain (hLOX-1ECD) in E.coli, establishment of conditions for the efficient soluble expression and establishment of large-scale large amount purification process. The efficient soluble expression and purification method can be used for preparing large amount of hLOX-1ECD. According to the efficient soluble expression and purification method of human LOX-1 extracellular domain, the purification process is simple, convenient and feasible, a large amount preparation can be realized, and the purified hLOX-1ECD protein has high purity.

Description

technical field [0001] The invention discloses a high-efficiency soluble expression and purification method of human LOX-1 extracellular domain, and relates to the construction of an expression vector for highly efficient and soluble expression of human LOX-1 extracellular domain (hLOX-1 ECD) in Escherichia coli. The establishment of soluble expression conditions and the establishment of a large-scale purification process can be used for the large-scale preparation of hLOX-1 ECD, which belongs to the technical field of bioengineering. Background technique [0002] The predecessor of cardiovascular and cerebrovascular diseases is atherosclerosis (Arteriosclerosis, hereinafter referred to as As). As is a disease that can lead to sudden disability and death after years of asymptomatic development, and 70%-80% of coronary thrombosis is secondary to the rupture of As plaques. In humans, hardening of the arteries with age is almost inevitable. As is reversible in the initial sta...

Claims

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Application Information

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IPC IPC(8): C12N15/63C12N15/12C07K14/705C07K1/22C07K1/18C07K1/16
Inventor 相宏宇谢秋宏孟兆丽张海辉张欣冉谢彦博
Owner JILIN UNIV
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