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Anti-apoptotic protein PTD-FNK protein and method for preparing same

A technology of PTD-FNK and anti-apoptotic protein, which is applied in the field of anti-apoptotic protein PTD-FNK protein and its preparation, can solve the problems of hindering the application process of protein, restricting the mass production of protein, and large protein loss, etc., and achieve the meticulous preparation method Rigorous, highly repeatable, and expressive effects

Inactive Publication Date: 2018-08-07
GUANGXI UNIV
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Problems solved by technology

[0003] PTD-Bcl-xL protein function-enhanced mutant PTD-FNK protein has a significant role in protecting cells against external adverse factors (low temperature, apoptosis inducers, lack of serum, etc.) The form is inclusion body, which needs to go through the renaturation process to obtain soluble protein, and the protein is greatly lost in the renaturation process, which seriously limits the mass production of protein and hinders the application process of protein in production practice

Method used

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  • Anti-apoptotic protein PTD-FNK protein and method for preparing same
  • Anti-apoptotic protein PTD-FNK protein and method for preparing same
  • Anti-apoptotic protein PTD-FNK protein and method for preparing same

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Embodiment

[0044] A preparation method of anti-apoptotic protein PTD-FNK protein, comprising the following steps:

[0045] (1) Take the fresh liver tissue of SD rats, and extract the total RNA according to the instructions of the RNA isolater reagent;

[0046] (2) Construction of the cloning plasmid pUM19-T-Bcl-xL;

[0047] (3) Construct the prokaryotic expression plasmid pET28a(+)-PTD-Bcl-xL;

[0048] (4) Site-directed mutation of the prokaryotic expression plasmid pET28a(+)-PTD-Bcl-xL to obtain the recombinant plasmid pET28a(+)-PTD-FNK plasmid;

[0049] (5) Induce the expression of the fusion protein PTD-FNK in BL21 competent cells, and induce for 14 hours at 30°C;

[0050] (6) Collect bacteria, ultrasonically crush, power 400W, crush for 30min, sonicate for 2S, pause for 6S as a cycle, centrifuge for the first time, 12000rpm, 4°C, centrifuge for 20min, collect supernatant, and use inclusion body washing solution for precipitation ( 10mmol / L Tris-HCL, 50mmol / L NaCl, 2mol / L urea) twi...

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Abstract

The invention provides an anti-apoptotic PTD-FNK protein and a method for preparing the same. Total RNA (ribonucleic acid) of the livers of rats is used as a template, prokaryotic expression vector pET28a(+)-PTD-FNK plasmids of PTD-FNK proteins are constructed by the aid of molecular cloning technologies and gene point mutation technologies, the prokaryotic expression vector pET28a(+)-PTD-FNK plasmids are transformed into Escherichia coli BL21(DE3), PTD-FNK soluble expression is induced at the low temperatures, and proteins are extracted from supernatants and precipitates.

Description

technical field [0001] The invention relates to an anti-apoptosis protein PTD-FNK protein and a preparation method thereof. Background technique [0002] Bcl-xL protein is considered to be a potential therapeutic protein for abnormal apoptosis. However, once Bcl-xL is phosphorylated, its anti-apoptotic activity will be inhibited, which limits its therapeutic efficacy. Therefore, Makoto Sakurazawa et al. observed the crystal structure of rat Bcl-xL in 2000, and mutated 3 amino acids of Bcl-xL (tyrosine at 22 to phenylalanine (F), glutamine at 26 Mutated to asparagine (N) and arginine at position 165 to lysine (K) to construct a super anti-apoptotic factor FNK (originally named bcl-x FNK), becoming the first mammalian apoptotic factor The first and only mutant with enhanced function. Transfection of FNK cDNA into cells protects against various stimuli, including oxidative stress (hydrogen peroxide and paraquat), Ca 2+ Vector (A23187), growth factor depletion (serum and IL-3)...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K19/00C12N15/70
CPCC07K14/4747C07K2319/10C12N15/70
Inventor 李珣王晓晔胡传活石博妹李丹丹刘蛟陈正赵艳
Owner GUANGXI UNIV
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