Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A method for expressing biotinylated luciferase

An expression method, luciferase technology, which is applied in the field of expression of biotinylated DNA polymerase, can solve the problems of low ATPS activity of fusion protein, reduce the expression level of fusion protein, and cannot better meet the practical application, so as to increase the yield , the effect of activity improvement

Active Publication Date: 2019-05-31
BEIJING ZHONGKEZIXIN TECH
View PDF0 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the biological modification method can avoid the disadvantages of the chemical modification method, when the recombinant expression vector constructed by using BCCP87 and ATPS is used for fusion expression, since 87 more amino acid residues need to be expressed, the expression level of the fusion protein is reduced to a certain extent; and there are Recombinant protein expressed in partial inclusion bodies; the ATPS activity of the expressed fusion protein is relatively low
Therefore, it cannot better meet the practical application

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • A method for expressing biotinylated luciferase
  • A method for expressing biotinylated luciferase
  • A method for expressing biotinylated luciferase

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Embodiment 1 Expression method of biotinylated luciferase

[0036] Include the following steps:

[0037] ⑴. Select the North American firefly luciferase whose GenBank number is CAA46407, and design a pair of specific primers: the nucleotide sequence of the forward primer is shown in SEQ ID NO: 3, and the restriction site is BamHI; The nucleotide sequence is shown in SEQ ID NO: 4, and the enzyme cutting site is XhoI;

[0038] (2) The forward primer and reverse primer described in step (1) of chemical synthesis embodiment 1, with - luc vector as a template, amplify the fusion coding gene of the biotin acceptor peptide of the present invention and the luciferase described in step (1) of Example 1 by PCR, the PCR program is: denaturation at 95°C for 10 minutes, denaturation at 95°C for 30s , anneal at 50°C for 30s, extend at 72°C for 1.5min, 35 cycles, and finally extend at 72°C for 10min;

[0039] ⑶. The DNA gel recovery kit reclaims the fusion coding gene obtained in ...

Embodiment 2

[0042] Embodiment 2 Purification of biotinylated luciferase

[0043] The biotinylated luciferase expressed according to the method of Example 1 was purified using a histidine affinity chromatography column.

[0044] The purification method comprises the following steps:

[0045] ⑴. Centrifuge to collect the bacterial cells after the induction culture in step ⑸ of Example 1, crush the bacterial cells by ultrasonic waves, centrifuge the crushed liquid at 4°C and 12000rpm / min for 30min, and collect the supernatant;

[0046] ⑵. Equilibrate the histidine affinity chromatography column with 5 times the volume of the column bed (50mM pH8.0 sodium phosphate buffer + 300mM sodium chloride + 5mM imidazole);

[0047] (3) The supernatant obtained in step (1) of Example 2 was added to a histidine affinity chromatography column at a flow rate of 1 mL / min, so that biotinylated luciferase was attached to the column;

[0048] ⑷. Rinse the chromatography column with 15 times the volume of the...

Embodiment 3

[0051] Embodiment 3 Expression method of biotinylated luciferase

[0052] Include the following steps:

[0053] ⑴. Select a heat-resistant mutant rt-LlL of Japanese firefly luciferase, which is replaced by leucine by replacing 217 alanine (Ala) in Japanese firefly luciferase with GenBank numbering X66919.1 acid (Leu) acquisition (Xu Shu, Zou Bingjie et al. Expression of thermostable luciferase and establishment of thermostable pyrosequencing system[J]. Acta Biological Engineering, 2012, 06:763-771); design a pair of specific Sexual primer: the nucleotide sequence of the forward primer is shown in SEQ ID NO: 5, and the restriction site is BamHI; the nucleotide sequence of the reverse primer is shown in SEQ ID NO: 6, and the restriction site is HindⅢ ;

[0054] (2) The forward primer and the reverse primer described in Step (1) of Example 3 were chemically synthesized, and the rt-L1L recombinant vector containing the coding gene of the heat-resistant mutant rt-L1L described in...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

The invention provides an expressing method for biotinylation luciferase. The expressing method includes the steps that biotin receptor peptide GLNDIFEAQKIEWH composed of fourteen amino acid residues is used, encoding genes of the biotin receptor peptide and luciferase encoding genes are fused and expressed in escherichia coli host cells, and expressed fusion protein can be modified in a biotinylation mode in the escherichia coli host cells. Compared with the technology that biotinylation modification is carried out through BCCP87, the expressing method has the advantages that the yield of the biotinylation luciferase is increased by 47% or above, and the catalytic activity is improved by 10 times or above. After Bead-LUC obtained after the biotinylation luciferase expressed according to the expressing method is fixed through a magnetic bead is repeatedly washed 10 times and 15 times, 93 % or above of the relative activity and 90% or above of the relative activity generated before washing can still be kept.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to an expression method of biotinylated DNA polymerase. Background technique [0002] Firefly luciferase (EC 1.13.12.5-7) is an oxidoreductase. Firefly luciferase in adenosine triphosphate (ATP), O 2 and Mg 2+ With the participation of D-fluorescein, it can catalyze the oxidative decarboxylation of D-fluorescein, convert chemical energy into light energy, and release yellow-green fluorescence at the same time. The specific reaction process and luminescent mechanism are as follows: firefly luciferase first converts luciferin and Mg-ATP complex into the corresponding transition state product luciferin adenylate; the transition state product rapidly reacts with O 2 After the reaction, adenosine monophosphate (AMP) is released, and the excited state product oxidized luciferin is formed; the excited state product releases light quanta and returns to the ground state. The most...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/02C12N15/70C12N15/62
CPCC07K2319/61C12Y113/12007
Inventor 高静蔡亦梅吴超徐潇张睿王者馥王绪敏殷金龙任鲁风
Owner BEIJING ZHONGKEZIXIN TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com