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Method and special expression vector thereof for promoting solubility expression of foreign protein by TAT (Trans-activator transduction) core peptide

A technology of exogenous protein and expression vector, which is applied in the application field of biotechnology products, can solve the problems of staying and no data to introduce TAT polypeptide research, etc., and achieve the effect of promoting high efficiency, saving cost, and increasing expression

Inactive Publication Date: 2014-04-02
INST OF RADIATION MEDICINE ACAD OF MILITARY MEDICAL SCI OF THE PLA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Method and special expression vector thereof for promoting solubility expression of foreign protein by TAT (Trans-activator transduction) core peptide
  • Method and special expression vector thereof for promoting solubility expression of foreign protein by TAT (Trans-activator transduction) core peptide
  • Method and special expression vector thereof for promoting solubility expression of foreign protein by TAT (Trans-activator transduction) core peptide

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0038] 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

[0039]First, the core coding sequence fragment of the double-stranded TAT containing the enzyme cleavage sites NcoI (upstream) and NdeI (downstream) is synthesized by chemical synthesis (select sequence 2 in the sequence list in this embodiment), and then use restriction endonucleation The core fragment and the empty vector pET28b were double-digested with enzymes NcoI and NdeI, and the target fragments were recovered separately, then the exogenous DNA fragment and the empty vector were ligated by T4 DNA 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 pET28b-TAT conta...

Embodiment 2

[0040] Example 2. Using TAT core peptide to promote efficient and soluble expression of enhanced green fluorescent protein (EGFP)

[0041] 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:

[0042] 1. Construction of prokaryotic expression vectors pET28b-TAT-EGFP and pET28b-EGFP carrying the gene encoding enhanced green fluorescent protein (EGFP)

[0043] 1. Amplify the target gene EGFP by polymerase chain reaction (PCR)

[0044] 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 O 38.25 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

[0060] Example 3. Utilizing the TAT core peptide to promote efficient and soluble expression of human neuroglobin (rhNGB)

[0061] 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:

[0062] 1. Construction of prokaryotic expression vectors pET28b-TAT-NGB and pET28b-NGB carrying the gene encoding human neuroglobin (rhNGB)

[0063] 1. Amplify the target gene NGB by polymerase chain reaction (PCR)

[0064] The cDNA fragment of NGB was amplified by conventional PCR method, and 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 O 38.25 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 ...

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Abstract

The invention discloses a method and a special expression vector thereof for promoting a prokaryotic expression system to efficiently perform solubility expression of foreign protein by TAT (Trans-activator transduction) core peptide. The special expression vector is a prokaryotic expression vector containing one or more TAT core polypeptide coding regions. The method comprises the following steps of: (1) inserting foreign genes into the special expression vector to obtain the prokaryotic expression vector containing TAT core polypeptide coding regions and foreign genes; (2) converting the prokaryotic expression vector into the prokaryotic expression system; (3) performing inducible expression of the prokaryotic expression vector; and (4) preparing foreign protein. Proven by detection, the expression product mainly exists in a soluble form in host bacteria lysis supernatant, and accounts for 25-30% of the total protein in the lysis supernatant, so that high yield foreign protein can be obtained and a big amount of foreign protein with biological activity can be prepared.

Description

technical field [0001] The invention belongs to the application field of biotechnology products, and relates to a large-scale preparation method of exogenous proteins, in particular to a method for promoting efficient and soluble expression of exogenous proteins by using TAT core peptides and a special expression carrier thereof. Background technique [0002] 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 large-scale preparation of functional and highly active proteins and further significantly improve the production efficiency of biological products is not only an urgent problem in the field of bioengineering, but also a major problem in the field of protein-based drug research and development. [0003] TAT (Trans-activator transduction) is a polypeptide rich in basic amino acid sequence encoded by HIV-1 (Human Immunodeficiency Virus-1), which be...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/70C12N15/75C12N15/67C12N15/49C12P21/00
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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