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Expression and purification method for bombyx mori odorant binding protein (BmOBP2)

A technology for expression and purification of odorant binding protein, which is applied in the field of expression and purification of silkworm odorant binding protein BmOBP2, which can solve the problems of strong hydrophobicity, low abundance, and microscopic inhomogeneity, and achieve the effect of solving low expression level

Inactive Publication Date: 2015-04-15
TIANJIN YAOYU BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although transmembrane proteins have such important biological functions, due to their low abundance, strong hydrophobicity, and microscopic heterogeneity, it is very difficult to separate, purify, and crystallize native membrane proteins

Method used

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  • Expression and purification method for bombyx mori odorant binding protein (BmOBP2)
  • Expression and purification method for bombyx mori odorant binding protein (BmOBP2)
  • Expression and purification method for bombyx mori odorant binding protein (BmOBP2)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0049] Example 1: Primer Design

[0050] Amplify the BmOBP2 target gene, the gene sequence is shown in SEQ ID NO.1, and the primers are designed as follows:

[0051] Upstream primer F: 5’- CGC GGATCC ATGAAGAGCAAAACAAAAC-3' (underlined restriction endonuclease Bam H I restriction site), as shown in SEQ ID NO.3;

[0052] Downstream primer R: 5'- CCG CTCGAG TTATAGTTCATCTTTAAC-3' (underlined restriction endonuclease xho I restriction site), as shown in SEQ ID NO.4;

Embodiment 2

[0053] Example 2: Construction of pET-32a-BmOBP2 recombinant expression vector

[0054] (1) Using the silkworm chrysalis cDNA as a template, use the primers F and R designed in Example 1 to amplify the specific fragment. The specific procedures are: pre-denaturation at 94°C for 5 minutes, denaturation at 94°C for 1 minute, annealing at 62°C for 45 seconds, and extension at 72°C 30s, cycled 30 times; finally extended at 72°C for 10 min, recovered product to obtain BmOBP2 gene (see figure 1 ), its nucleotide sequence is shown in SEQ ID NO.1.

[0055] In a 50 μL centrifuge tube, add the following components:

[0056] wxya 2 O 32.5 μL

[0057] TAQ Buffer 5μL

[0058] 2mM dNTPs 5μL

[0059] F 1.5μL

[0060] R 1.5 μL

[0061] cDNA 3μL

[0062] TAQ Polymerase (2U / μL) 1.5μL

[0063] After mixing the components evenly, put them into the PCR machine, and design 30 cycles according to the above reaction parameters. After the reaction is over, identify the amplified fragment by...

Embodiment 3

[0065] Embodiment 3: Expression of recombinant protein

[0066] The expression vector recombined in Example 2 was transformed into E.coli BL21 (purchased from Fermentas Company) to obtain the recombinant bacteria, in LB medium containing 50 mg / mL ampicillin (Shanghai Hengyuan Biotechnology Co., Ltd.) (purchased from Oxoid company) at 37°C, shaking at 220 rpm until OD 600 ≈0.5, add isopropyl-B-D-thiogalactopyranoside (IPTG) (final concentration 1 mM), induce culture at 37°C for 5 hours, take 1.5mL of bacterial liquid and centrifuge at 12000rpm, add 50μL of 20mM trimethylol Resuspend in aminomethane (Tris) (pH 8.0), then take 40 μL, add 10 μL 5× sample buffer, cook at 100°C for 10 minutes, and then centrifuge at 12000 rpm for 5 minutes to obtain recombinant protein samples, take 15 μL for SDS-PAGE, the results Such as Figure 3A As shown, it shows that the recombinant protein containing the HIS tag is successfully expressed by the above method and the expression level is very ...

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Abstract

The invention relates to an expression and purification method for bombyx mori odorant binding protein (BmOBP2). By designing a primer, an expression vector Pet-32a-BmOBP2 is constructed and recombined, a great quantity of recombined interest protein is obtained through inducible expression, and high-purity recombined protein can be obtained by carrying out two simple purification technologies namely nickel ion affinity chromatography and anion displacement chromatography on the recombined protein, so that the method lays a very important foundation for research of the three dimensional structure and biological function of the recombined protein, and the problem that the natural membrane protein has low expression quantity and is difficult to purify is solved.

Description

technical field [0001] The invention relates to a method for expressing and purifying silkworm odor-binding protein BmOBP2, which belongs to the technical field of genetic engineering proteins. Background technique [0002] Membrane proteins not only participate in and regulate various metabolic activities of cells, but also serve as a bridge for material energy exchange and signal transmission between cells and the outside world. The vast majority of diseases are caused by the defect of a specific membrane protein, and 80% of the drugs on the market now work by binding to the membrane protein. Although transmembrane proteins have such important biological functions, due to their low abundance, strong hydrophobicity, and microscopic heterogeneity, it is very difficult to separate, purify, and crystallize natural membrane proteins. Almost all OBPs contain 6 conserved cysteines, connected by 3 disulfide bridges, which reinforce the structure of OBPs. Since OBPs do not underg...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C07K14/435C07K1/36C07K1/34C07K1/22C07K1/18
Inventor 张耀洲刘玲玲杜瑶瑶陈剑清舒特俊
Owner TIANJIN YAOYU BIOLOGICAL TECH
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