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Application of TAT core peptide fragment in preparing efficiently and solubly expressed exogenous protein

An exogenous protein, soluble technology, applied in the application field of biotechnology products, can solve the problems of staying, no data to introduce TAT polypeptide research, etc., and achieve the effect of promoting high efficiency, saving costs and wide application prospects.

Inactive Publication Date: 2014-06-18
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But so far, all the researches have only stayed at the level of TAT-mediated transmembrane transduction of exogenous substances, and there is no information on the research on other functions of TAT polypeptides

Method used

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  • Application of TAT core peptide fragment in preparing efficiently and solubly expressed exogenous protein
  • Application of TAT core peptide fragment in preparing efficiently and solubly expressed exogenous protein
  • Application of TAT core peptide fragment in preparing efficiently and solubly expressed exogenous protein

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Example 1. Construction of a special expression vector pET28b-TAT that utilizes the TAT core peptide to promote efficient and soluble expression of foreign proteins

[0042] First, the core coding sequence fragment of the double-stranded TAT containing the enzyme cleavage sites NcoI (upstream) and NdeI (downstream) was synthesized by chemical synthesis (in this embodiment, sequence 2 in the sequence list was selected), and then restriction endonuclease was used to The core fragment and the empty vector pET28b were double-digested with enzymes NcoI and NdeI, and the target fragments were recovered respectively, then the exogenous DNA fragment and the empty vector were ligated by T4DNA ligase, and transformed into competent cells BL21(DE3). Plasmid DNA was extracted according to conventional methods and identified by NcoI and NdeI double enzyme digestion. Finally, it was sent to Yingjun Company for direct sequencing. It was confirmed that the prokaryotic expression vector ...

Embodiment 2

[0043] Example 2. Using TAT core peptide to promote efficient and soluble expression of enhanced green fluorescent protein (EGFP)

[0044] Such as figure 1 As shown, promote efficient, soluble expression of enhanced green fluorescent protein (EGFP) with the method of the present invention, and specific method comprises the following steps:

[0045] 1. Construction of prokaryotic expression vectors pET28b-TAT-EGFP and pET28b-EGFP carrying the gene encoding enhanced green fluorescent protein (EGFP)

[0046] 1. Amplify the target gene EGFP by polymerase chain reaction (PCR)

[0047] The cDNA fragment of EGFP was amplified by conventional PCR method. The 50 μl PCR reaction system was: template DNA 0.5 μl; 10×dNTP 5 μl; 10×LA Taq buffer 5 μl; upstream and downstream primers 0.5 μl; LA Taq enzyme 0.25 μl, ddH 2 O38.25 μl. The PCR reaction conditions are: 95°C for 2 minutes; 95°C for 30 seconds, 56°C for 45 seconds, 72°C for 60 seconds (32 cycles); 72°C for 7 minutes. After the ...

Embodiment 3

[0063] Example 3. Utilizing the TAT core peptide to promote efficient and soluble expression of human neuroglobin (rhNGB)

[0064] Such as figure 1 As shown, the method of the present invention is used to promote the efficient and soluble expression of human source neuroglobin (rhNGB), and the specific method comprises the following steps:

[0065] 1. Construction of prokaryotic expression vectors pET28b-TAT-NGB and pET28b-NGB carrying the gene encoding human neuroglobin (rhNGB)

[0066] 1. Amplify the target gene NGB by polymerase chain reaction (PCR)

[0067] The cDNA fragment of NGB was amplified by the conventional PCR method. The 50 μl PCR reaction system was: 0.5 μl of plasmid template; 5 μl of 10×dNTP; 5 μl of 10×Ex Taq buffer; 0.5 μl of upstream and downstream primers; 0.25 μl of Ex Taq enzyme, ddH 2 O38.25 μl. The PCR reaction conditions are: 95°C for 4 minutes; 95°C for 45 seconds, 56°C for 30 seconds, 72°C for 45 seconds (30 cycles); 72°C for 7 minutes. After t...

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Abstract

The invention relates to an application of a TAT core peptide fragment in preparing an efficiently and solubly expressed exogenous protein and discloses a method for efficiently and solubly expressing the exogenous protein by using a prokaryotic expression system under promotion of the TAT core peptide fragment, and a special expression vector. By implementation of the method and the special expression vector, not only can the soluble exogenous protein be obtained, but also the exogenous protein with high yield can be obtained, so that a foundation is laid and a key technology is provided for preparing the exogenous protein with bioactivity on large scale, and the problem that the exogenous protein is difficult to efficiently express in the fields of bioengineering, research and development of drugs and the like is solved, and thus the cost of separating and purifying the exogenous protein in the following steps can be remarkably saved.

Description

[0001] The present invention is a divisional application of the patent application with application date of March 8, 2010, application number 201010119397.8, and the title of the invention is "Method for Promoting Soluble Expression of Foreign Protein by Using TAT Core Peptide and Its Special Expression Vector". technical field [0002] The invention belongs to the field of application of biotechnology products, and relates to a method for preparing a large amount of exogenous proteins, in particular to the application of TAT core peptides in the preparation of highly efficient and soluble expressed exogenous proteins and a method of using TAT core peptides to promote high-efficiency exogenous proteins. , The method of soluble expression and its special expression vector. Background technique [0003] As we all know, protein is an important executor of the body's life activities, and it is also the material basis of protein drugs widely used in the market. How to realize the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/70C12N15/75C07K14/155
Inventor 张成岗吴永红高艳
Owner INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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